WO2008047799A1 - Réactif d'analyse immunologique et procédé d'analyse immunologique - Google Patents
Réactif d'analyse immunologique et procédé d'analyse immunologique Download PDFInfo
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- WO2008047799A1 WO2008047799A1 PCT/JP2007/070190 JP2007070190W WO2008047799A1 WO 2008047799 A1 WO2008047799 A1 WO 2008047799A1 JP 2007070190 W JP2007070190 W JP 2007070190W WO 2008047799 A1 WO2008047799 A1 WO 2008047799A1
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- antibody
- latex
- antigen
- latex particles
- reagent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Definitions
- the present invention relates to an immunological analysis reagent and an immunological analysis method.
- the “analysis” in the present specification includes both “measurement” for quantitatively or semi-quantitatively determining the amount of the analyte, and “detection” for determining the presence or absence of the analyte. Is included.
- Latex agglutination is a method for measuring analytes in a short time by visually or optically detecting the degree of agglomeration of latex particles caused by the reaction between analytes and antigens or antibodies bound to latex particles. This is how to do it.
- the analyte is trapped by the first antibody bound to the magnetic particles, collected with a magnet, and washed with unreacted materials (so-called B / F separation), then the enzyme A sandwich method is also frequently used, in which a second antibody labeled with a signal-generating substance such as a fluorescent agent is added and analyzed.
- a signal-generating substance such as a fluorescent agent
- the ability to immobilize antigens or antibodies directly on latex particles by physical adsorption, or functional groups on the surface of latex particles, such as amino groups, carboxyl groups, mercapto groups, hydroxyl groups, aldehyde groups, or A method of binding an antigen or antibody to latex particles by covalent bonding via an epoxy group or the like has been used.
- Non-Patent Document 1 As a new problem in recent years, as disclosed in Non-Patent Document 1, antigen or By binding the antibody, structural denaturation of the antigen or antibody occurred, and there were cases in which a sample causing a non-specific reaction due to a “substance binding to the denaturation site” coexisting in the sample was observed. As a result, the reliability of the measurement results is lost, and accurate disease diagnosis cannot be performed. This problem is a very difficult problem that cannot be solved by examining individual antigens, antibodies, or latex particles.
- Non-Patent Document 1 “Clinical Pathology”, August 2000, Vol. 48, No. 8, 760-763
- latex particles produced by low-temperature miniemulsion polymerization that coexist with antigens or antibodies when synthesizing latex particles can be washed without conventional binding by introducing antigens or antibodies onto the surface of latex particles when synthesis is completed. It was possible to produce latex particles that were not removed by the treatment, and that antigens or antibodies bound by centrifugation or dispersion treatment were not peeled off. High reactivity can be obtained by using the above particles.
- latex particles produced by miniemulsion polymerization that coexist with antigen or antibody when synthesizing latex particles are introduced due to steric hindrance due to binding because the antigen or antibody is introduced to the latex particle surface when synthesis is completed.
- denaturation does not occur, and it has become possible to avoid reactions with specimens that cause non-specific reactions, which were previously observed.
- the present invention is based on these findings.
- an object of the present invention is to provide an immunological analysis reagent and an immunological analysis method that have high detection sensitivity and can avoid non-specific reactions that have conventionally occurred. Me ⁇ ⁇ .
- the problem is that, in the synthesis reaction of latex particles according to the present invention, monomers are polymerized in a state where antigens or antibodies to the analyte are present, and the antigens or antibodies are introduced onto the surfaces of the latex particles.
- This can be solved by immunological analysis reagents, including suspensions of latex particles.
- the present invention also includes (1) a test sample that may contain an analysis target compound, and (2) in the synthesis reaction of latex particles, the monomer is polymerized in the state in which an antigen or an antibody against the analysis target substance coexists. Latex particles having the antigen or antibody introduced on the surface of the latex particles;
- the present invention relates to an immunological analysis method in which a solution is contacted in a liquid.
- the antigen-antibody reaction occurs after the contact.
- the degree of aggregation of latex particles is optically analyzed.
- the liquid and the latex particles are separated, and the analysis target substance bound to the latex particles or the analysis target substance remaining in the liquid is added. analyse.
- the present invention provides:
- a polymer fine particle is produced in the presence of an antigen or an antibody.
- mini-emulsion polymerization power is a low-temperature emulsion polymerization in which the polymerization reaction is performed at a low temperature
- the emulsifier is a polymerizable surfactant having a polyethylene glycol chain.
- Latex particles produced by low-temperature miniemulsion polymerization that generates antigens or antibodies when synthesizing latex particles can be removed by washing without being bound conventionally by introducing antigens or antibodies to the surface of the latex particles when synthesis is completed. Centrifuge or Latex particles can be prepared in which the antigen or antibody bound by the dispersion treatment does not peel off, and high reactivity can be obtained by using such latex particles.
- the antigen or antibody is introduced to the latex particle surface when the synthesis is completed, denaturation due to steric hindrance due to binding does not occur, and thus avoids the reaction with a specimen that causes a non-specific reaction that has been conventionally observed. Can do.
- FIG. 1 shows the results of measuring a 2-fold dilution series of an anti-BSA antibody standard solution using the reagent of the present invention using BSA-introduced latex particles and the conventional reagent using BSA-bound latex particles. It is a graph.
- FIG. 2 is a graph showing the results of measuring a 2-fold dilution series of an anti-IgG antibody standard solution using the reagent of the present invention using BSA-introduced latex particles and a conventional reagent using BSA-bound latex particles. .
- Latex particles instead of the conventionally used latex particles having antigens or antibodies supported on the latex surface by physical or chemical bonding, antigens or antibodies (that is, immunology) on the surface of the latex particles are used.
- Latex particles (hereinafter referred to as partner-introduced latex particles).
- latex particles for example, the ability to directly immobilize antigens or antibodies on the latex particles by physical adsorption, or functional groups on the surface of the latex particles, such as amino groups, carboxyl groups, mercapto groups, hydroxyl groups.
- antigens or antibodies were bound to latex particles by covalent bonds via aldehyde groups or epoxy groups.
- the partner-introduced latex particles used in the present invention are not particularly limited as long as they are latex particles in which a part of the antigen or antibody is embedded in the latex particle so that the antigen or antibody is supported on the latex surface. It is not something.
- Such partner-introduced latex particles can be prepared, for example, by the production method of the present invention.
- miniemmanoreaction polymerization for example, M. Antonietti,. Landfester, Prog. Polym. Sci.
- miniemmanoreaction polymerization for example, M. Antonietti,. Landfester, Prog. Polym. Sci.
- an antigen or an antibody coexists in the polymerization reaction.
- 2002, 27, 689-757 J. M. Asua, Prog. Polym. Sci., 2002, 27, 1283-1346.
- Normal miniemulsion polymerization is not limited to this, for example, a step of mixing a monomer, a radical polymerization initiator, an emulsifier, and a hydrophobe, a step of shearing the mixture
- the method may include the step of polymerizing the mixture by heating to the polymerization start temperature.
- miniemulsion polymerization after the polymerization monomer and the emulsifier are mixed, for example, by carrying out a shearing process by ultrasonic irradiation, the monomer is torn off by the shearing force, and monomer fine oil droplets covered with the emulsifier are formed. It is formed.
- the monomer fine oil droplets are polymerized as they are to obtain polymer fine particles.
- the antigen or antibody used in the present invention is not particularly limited as long as it is a surface-active antigen or antibody, and is used for a latex method (for example, latex agglutination method, B / F separation using latex).
- a possible antigen or antibody is preferred. Whether or not a certain antigen or antibody exhibits surface activity can be confirmed by a known method, for example, surface tension measurement or fluorescence spectrum measurement using pyrene as a fluorescent probe.
- the intensity of the first emission peak (II) and the third emission peak (13) of pyrene is used for measurement of fluorescence spectrum using the pyrene as a fluorescent probe.
- Antigens or antibodies with a ratio (I1 / I3) of 0.5 to; 1.6 (more preferably 0.6 to 1.5) are preferred.
- the antigen or antibody can include various antibodies, receptors, enzymes, lipids, sugar chains, and the like.
- IgG C-reactive protein
- CRP C-reactive protein
- ferritin ferritin
- ⁇ -2 micro globulin alpha - Hue preparative protein
- AFP alpha - Hue preparative protein
- IgE B hepatitis virus
- D dimer fibrin 'fibrinogen degradation products
- FDP fibrin 'fibrinogen degradation products
- soluble Fuiburi emissions soluble fibrin: SF
- PPI plasmin ⁇ ⁇ 2-plasmin inhibitor complex
- PSA prostate specific antigen
- elastase 1 elastase XDP
- thrombomodulin or albumin (preferably serum albumin).
- a monoclonal antibody or a polyclonal antibody can be used.
- the kind of the antibody the power of the immunoglobulin molecule itself, an antibody fragment such as Fab, Fab ′, F (ab ′) or Fv can be used.
- an antibody fragment such as Fab, Fab ′, F (ab ′) or Fv can be used.
- a monomer that can be used in the present invention a monomer that can be used in ordinary miniemulsion polymerization can be used.
- styrene for example, chloromethylstyrene, sodium styrenesulfonate
- Acrylic acid or methacrylic acid acrylic acid ester or methacrylic acid ester [for example, methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, hexadecyl (meth) acrylate), butyl acetate
- Acrylic acid or methacrylic acid acrylic acid ester or methacrylic acid ester [for example, methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, hexadecyl (meth) acrylate), butyl acetate
- acrylic acid ester or methacrylic acid ester for example, methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, hexadecyl (me
- radical polymerization initiator that can be used in the present invention
- a radical polymerization initiator that can be used in ordinary miniemulsion polymerization can be used.
- a peroxide initiator, a persulfuric acid initiator examples include azo initiators and redox initiators.
- the antigen or antibody coexisting in the polymerization reaction has physiological activity, the decrease in physiological activity can be suppressed by performing the polymerization reaction at a low temperature.
- Examples of the peroxide initiator include benzoyl peroxide (BPO), di-tert-butyl peroxide (DBPO), and ammonium peroxide.
- Examples of the persulfate initiator include potassium persulfate (KPS), ammonium persulfate (APS), and sodium persulfate (NPS).
- Examples of the azo-based initiator include azobisisobutyronitrinole (AIBN), dimethinole 2, 2, monoazobisisobutyrate (MAIB), 4, 4, monoazobis (4-cyananovaleric acid), 2, 2, -Mentioning azobis (2,4-dimethylvaleronitrile).
- the redox initiator for example, N, N, N ', N'-tetramethyl ethylene ⁇ Min [ ⁇ , ⁇ , ⁇ ', ⁇ '- tetramethylethylenediamine (TMEDA)] / over 3 ⁇ 4f potassium L acid [potassiu m
- KPS persulfate
- FeSO / KPS FeSO / H 2 O
- emulsifier that can be used in the present invention
- various surfactants generally used in miniemulsion polymerization can be used, and examples thereof include an anionic compound, a cationic compound, and a nonionic compound. be able to.
- the nonionic compound include Examples thereof include a polymerizable surfactant having a polyethylene glycol (PEG) chain, a long-chain alcohol, a polybulu alcohol, and Brij 35 (PIERCE).
- Examples of the long-chain alcohol include 1 pentanol and decanol.
- hydrophores that can be used in ordinary miniemulsion polymerization can be used.
- hexadecane, silsesquioxane, or a hydrophobic polymer for example, , Polystyrene, polymethyl methacrylate
- hexadecane or polystyrene is preferred.
- hydrophobes it is possible to suppress the increase in particle size non-uniformity due to Ostwald ripening and to synthesize monodisperse latex particles.
- the reaction conditions at the time of polymerization are the types of monomers and antigens or antibodies used, the average particle diameter of the polymer fine particles to be synthesized, and the antigen supported on the fine particle surface. Alternatively, depending on the amount of antibody, etc., it can be appropriately determined by conducting a pilot test, for example.
- an antigen or an antibody for example, ushi serum albumin, IgG, F (ab ′) is usually from 0.01 to 5 g, preferably
- radical polymerization initiator eg ascorbic acid, HO
- HO corbic acid
- An agent eg NK ester M-230G, NK ester M-90G
- An agent usually in an amount of 0.;! To 10 mmol, preferably 0.2 to 5 mmol, more preferably 0.4 to 4 mmol;
- the power to use is S.
- the reaction time is usually 1 hour or longer, preferably 3 to 48 hours, more preferably 4 to 24 hours.
- the reaction temperature is usually 0 to 80 ° C, preferably 0 to 60 ° C, more preferably 0 to 40 ° C.
- the mechanism of action in which the antigen or antibody coexisting during the miniemulsion polymerization is introduced onto the surface of the polymer fine particle (for example, latex) is not clear. I guess as follows. That is, since the antigen or antibody used in the present invention exhibits surface activity, it is considered that it exists on the surface of polymer fine particles (for example, latex) which is a bonding zone with water due to its hydrophilic portion. The present invention is not limited to this estimation.
- Latex method for example, latex agglutination method, B / F separation using latex
- a latex particle into which an antigen or antibody (for example, a polyclonal antibody or a monoclonal antibody, or an antibody fragment thereof) to the analyte is introduced is brought into contact with the analyte.
- the introduced antigen or antibody is in a state that is not denatured due to steric hindrance, it does not react with a non-specific sample for the denatured site, and analyzes the analyte with higher sensitivity than the conventional method (detection or measurement, Preferably).
- Substances (analytical substances) that can be analyzed according to the present invention are not particularly limited as long as they are substances (especially physiologically active substances) that can be analyzed as antigens or antibodies generally using an antigen-antibody reaction.
- Representative examples of the analysis target substance can include proteins, lipids, sugar chains and the like, and more specifically, for example, the ability to list various antibodies, receptors, enzymes, and the like.
- IgG C-reactive protein (CRP), ferritin, ⁇ -2 microglobulin, ⁇ -fetoprotein (AFP), IgE, hepatitis B virus (HBS antibody or HBc antibody), D dimer, fibrin 'Fibrinogen degradation product (FDP), soluble fibrin (SF), plasmin' ⁇ 2-plasmin inhibitor complex ( ⁇ ), prostate specific antigen (PSA), elastase 1, elastase XDP, thrombomodulin, or albumin (preferably And serum albumin).
- CRP C-reactive protein
- AFP ⁇ -2 microglobulin
- AFP ⁇ -2 microglobulin
- IgE hepatitis B virus
- HBS antibody or HBc antibody hepatitis B virus
- D dimer D dimer
- fibrin 'Fibrinogen degradation product FDP
- SF soluble fibrin
- SF plasmin' ⁇ 2-plasmin inhibitor complex
- PSA prostate specific antigen
- test sample that can be analyzed in the present invention is not particularly limited as long as it is a sample that may contain the analysis target substance.
- a biological sample such as blood, serum, plasma, urine, Examples thereof include cerebrospinal fluid, cell or tissue disruption fluid, and the like.
- a latex method is used in the analysis.
- latex particles into which an antigen or antibody that specifically reacts with the substance to be analyzed is introduced.
- the average particle diameter of the partner-introduced latex particles can be selected as appropriate depending on the detection concentration of the analyte or the measuring instrument, and is usually selected appropriately within the range of 0.05 to 0.5 m. .
- an antibody to be introduced into latex particles a monoclonal antibody or a polyclonal antibody can be used.
- an antibody fragment such as Fab, Fab ′, F (ab ′) or Fv can be used as the type of antibody.
- the immunological analysis reagent according to the present invention includes, for example, a one-part reagent containing latex particles and a buffer solution into which an antigen or antibody has been introduced; a first reagent that is a buffer solution, and an antigen or antibody introduced therein
- a two-component reagent consisting of a latex reagent and a second reagent containing both monoclonal antibodies; or sensitizing the antigen or antibody with the first reagent containing both buffer and introduced latex particles It can be in various forms such as a two-component reagent composed of a second reagent containing latex particles.
- the present invention by performing an agglutination reaction using the reagent as described above, and optically analyzing (particularly measuring) the degree of aggregation (aggregation degree) that has occurred, It is possible to analyze (especially measure) the amount of analyte in the test sample.
- measurement can be performed using an optical instrument that measures scattered light intensity, absorbance, or transmitted light intensity.
- a preferred measurement wavelength is 300 to 800 nm.
- the measurement method is to measure the increase or decrease in scattered light intensity, absorbance, or transmitted light intensity by selecting the size (average particle diameter) or concentration of latex particles to be used or setting the reaction time according to a known method. Can be done. It is also possible to use these methods in combination.
- the latex particles and the liquid are separated by performing B / F separation, and there is an analyte to be bound to the latex particles.
- Analyze (especially measure) the substance to be analyzed remaining in the liquid It is possible to analyze (particularly measure) the amount of the substance to be analyzed in the material.
- the latex particle agglutination reaction in addition to selecting the type of antigen or antibody to be introduced, is more precisely amplified by adjusting other factors that affect the latus status agglutination reaction, It is possible to further expand the quantifiable range in the low concentration range and further suppress non-specific reactions.
- Other factors that affect the latex agglutination reaction include the concentration of latex particles, the amount of antigen or antibody introduced into latex particles, or the particle size of latex particles.
- the reaction conditions for the antigen-antibody reaction used in the present invention may be the same as the normal conditions, and various buffer solutions may be appropriately selected according to the type of the substance to be analyzed.
- This buffer solution only needs to have an ion concentration or pH that does not inactivate the analyte and does not inhibit the antigen-antibody reaction.
- Good buffer, glycine buffer, or Tris buffer can be used.
- the pH of the reaction is preferably 5 to 10, particularly 6 to 8.
- the reaction temperature is preferably 0 to 50 ° C, particularly 20 to 40 ° C.
- the reaction time is determined by the power S.
- BSA-introduced latex particles The average particle diameters of the obtained BSA-introduced latex particles were 0 ⁇ 109 111 (when 30 °) and 0.121 111 (when 60 °).
- buffer A the NaC 1 -containing Tris buffer containing no BSA
- buffer B the NaCl-containing Tris buffer containing BSA
- a two-component system comprising the BSA-introduced latex particle suspension (second reagent) prepared in Example 1 (2) and buffer A or B (first reagent) prepared in Example 1 (3)
- second reagent prepared in Example 1
- buffer A or B first reagent
- Dilute anti-BSA antibody (Rabbit Anti cow albumin; DAKO, 900 units) twice with physiological saline, and add standard anti-BSA antibody dilution series at 900, 450, 225, 113, 56, 28, 14, and 7 unit concentrations. Prepared.
- Example 1 Mix 15 ml of the standard anti-BS A antibody dilution column with buffer solution ⁇ or ⁇ 90 ⁇ L prepared in Example 1 (3) and hold at 37 ° C for a while, and then prepare BSA prepared in Example 1 (2). 90 L of the introduced latex particle suspension was added and stirred, and then the absorbance at a wavelength of 800/570 nm after 5 minutes was measured. The difference between the change in absorbance at 570 nm and the change in absorbance at 800 nm was used as the change in absorbance ( ⁇ Abs). The measurement was performed using Hitachi automatic analyzer 7170 type.
- polystyrene latex particles having an average particle size of 0 ⁇ ll ⁇ m (manufactured by Nippon Synthetic Rubber: solid content of 10%) were used, and 0.3% (3 mg / A suspension of BSA-bound latex particles prepared by sensitizing the amount of (ml) was used.
- Table 1 The results are shown in Table 1, FIG. 1 and Table 2.
- Table 1 and Figure 1 use buffer A or B
- the results are shown together with the results of conventional reagents.
- Anti-B S A antibody Conventional reagent Reagent of the present invention
- the BSA-introduced latex particle suspension obtained by carrying out the polymerization reaction at a low temperature (30 ° C).
- the amount ( ⁇ Abs) of 8480 and the BSA-introduced latex particle suspension at low temperature were more sensitive.
- the absorbance change ( ⁇ Abs) is 385 in 900 units. there were.
- Example No. 1 to No. 5 normal human serum samples
- 3 non-specific human serum samples Sample No. 6 to The procedure of Example 2 (2) was repeated except that No. 8) was used.
- the non-specific specimen reacts non-specifically with the BSA-bound latex particle suspension in which BSA is bound to latex particles and binds BSA! /, NA! /, Latex particle suspension. Does not react! /, Is a sample
- IgG-introduced latex particles The average particle size of the IgG-introduced latex particles was 0 ⁇ 318 m.
- Blocking reagent for immunological measurement N101 (Nippon Yushi Co., Ltd.) lmL was added to IgG introduced latex particles (1% concentration) lmL, and stirred at room temperature for 30 minutes. This mixture was centrifuged at 35000 rpm. To the resulting precipitate, 10 mL of Tris buffer ( ⁇ 8.0) was added to suspend the latex to prepare an IgG-introduced latex particle suspension.
- a two-component reagent comprising the IgG-introduced latex particle suspension (second reagent) prepared in Example 4 (2) and buffer B (first reagent) prepared in Example 1 (3) was evaluated as an anti-IgG antibody measuring reagent as an immunological analysis reagent of the present invention in the following Examples.
- second reagent the IgG-introduced latex particle suspension
- buffer B first reagent
- Anti-IgG antibody (Anti-IgG H & L chains; MILES_YEDA, 2.8 mg / mL) was diluted twice with physiological saline to obtain 2.8, 1.4, 0.7, 0.35, 0.175, 0.00. 088 and 0.044 mg / m
- the IgG-introduced latex particle suspension 90 prepared in Example 4 (2) was added and stirred, and then the absorbance at a wavelength of 800/570 nm after 5 minutes was measured. The difference between the change in absorbance at 570 nm and the change in absorbance at 800 nm was used as the change in absorbance ( ⁇ Abs). The measurement was performed using Hitachi automatic analyzer 7170 type.
- polystyrene latex particles having an average particle size of 0.283 ⁇ m (Sekisui Co., Ltd .: solid content 10%) were used, and 1% IgG (1 Omg / mL) was added to 1 mL concentration of the particles. ) Quantity An IgG-bound latex particle suspension prepared by sensitization and a latex particle suspension without IgG sensitization were used.
- Anti-IgG antibody Reagent of the present invention Conventional reagent
- the average particle size of the soot particles was 0 ⁇ 249 am.
- Anti-CRP antibody F (ab ')-introduced latex particles (1% concentration) lmL immunoassay
- Blocking reagent N101 (Nippon Yushi Co., Ltd.) lmL was added and stirred at room temperature for 30 minutes. This mixture was centrifuged at 35000 rpm. Add 10 mL of Tris buffer (pH 8.0) to the resulting precipitate, suspend the latex, and anti-CRP antibody F (ab ')-introduced latex
- a particle suspension was prepared.
- Example 7 Measurement of CRP standard solution
- CRP (Oriental Yeast Co., Ltd. Recombinant CRP solution 100mg / dU diluted with physiological saline, 3.2, 1.6, 0.8, 0.4, 0.2, and ⁇ . Lmg / dL, 6 concentrations of standard CRP diluted ⁇ IJ) Prepared.
- the buffer solution 90 ⁇ L prepared in Example 1 (3) was mixed with 211 L of the standard CRP dilution series, and kept at 37 ° C for a timely time. Then, the anti-antibody 3 antibody prepared in Example 6 (2) (£ 1) Introduced latex particles
- the present invention can be applied to uses of immunological analysis (particularly latex agglutination method).
Description
Claims
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ES07829924T ES2702802T3 (es) | 2006-10-16 | 2007-10-16 | Reactivo y procedimiento de análisis inmunológico |
JP2008539827A JP5288349B2 (ja) | 2006-10-16 | 2007-10-16 | 免疫学的分析試薬及び免疫学的分析方法 |
EP07829924.5A EP2088429B1 (en) | 2006-10-16 | 2007-10-16 | Reagent and method for immunological analysis |
CN2007800385549A CN101573619B (zh) | 2006-10-16 | 2007-10-16 | 免疫学分析试剂和免疫学分析方法 |
US12/445,863 US20100197042A1 (en) | 2006-10-16 | 2007-10-16 | Reagent and method for immunological analysis |
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EP (1) | EP2088429B1 (ja) |
JP (1) | JP5288349B2 (ja) |
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US10557848B2 (en) | 2014-09-02 | 2020-02-11 | Lsi Medience Corporation | Polymer microparticle for carrying physiologically active substance and method for preparing same |
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CN102305857A (zh) * | 2011-08-02 | 2012-01-04 | 深圳市亚辉龙生物科技有限公司 | 一种测定幽门螺杆菌抗体的胶乳免疫试剂及其检测方法 |
CN111812336A (zh) * | 2020-08-10 | 2020-10-23 | 苏州康和顺医疗技术有限公司 | 用于检测冠状病毒抗体的检测试剂盒及其制备方法 |
CN113866412A (zh) * | 2021-09-07 | 2021-12-31 | 山东博科生物产业有限公司 | 一种灵敏的总前列腺特异性抗原检测试剂盒 |
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- 2007-10-16 US US12/445,863 patent/US20100197042A1/en not_active Abandoned
- 2007-10-16 JP JP2008539827A patent/JP5288349B2/ja not_active Expired - Fee Related
- 2007-10-16 WO PCT/JP2007/070190 patent/WO2008047799A1/ja active Application Filing
- 2007-10-16 ES ES07829924T patent/ES2702802T3/es active Active
- 2007-10-16 CN CN2007800385549A patent/CN101573619B/zh not_active Expired - Fee Related
- 2007-10-16 EP EP07829924.5A patent/EP2088429B1/en not_active Not-in-force
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Cited By (1)
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---|---|---|---|---|
US10557848B2 (en) | 2014-09-02 | 2020-02-11 | Lsi Medience Corporation | Polymer microparticle for carrying physiologically active substance and method for preparing same |
Also Published As
Publication number | Publication date |
---|---|
CN101573619A (zh) | 2009-11-04 |
JP5288349B2 (ja) | 2013-09-11 |
ES2702802T3 (es) | 2019-03-05 |
EP2088429A1 (en) | 2009-08-12 |
JPWO2008047799A1 (ja) | 2010-02-25 |
EP2088429A4 (en) | 2010-01-20 |
CN101573619B (zh) | 2013-05-08 |
EP2088429B1 (en) | 2018-10-03 |
US20100197042A1 (en) | 2010-08-05 |
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