WO2009123060A1 - 精製血清アルブミン及び免疫学的測定方法 - Google Patents
精製血清アルブミン及び免疫学的測定方法 Download PDFInfo
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- WO2009123060A1 WO2009123060A1 PCT/JP2009/056316 JP2009056316W WO2009123060A1 WO 2009123060 A1 WO2009123060 A1 WO 2009123060A1 JP 2009056316 W JP2009056316 W JP 2009056316W WO 2009123060 A1 WO2009123060 A1 WO 2009123060A1
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- serum albumin
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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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- the present invention relates to purified serum albumin having a small lot difference and an immunoassay method having high reactivity and less non-specific reaction using the purified serum albumin.
- an immunological measurement method As a method for measuring trace substances contained in blood, urine and the like, an immunological measurement method is employed.
- the immunological measurement method is based on specific strong binding of an antigen-antibody reaction, and can specifically measure a target substance with high sensitivity even from a sample in which various substances are mixed.
- trace components such as cancer markers in blood
- antigens such as viruses, antibodies against bacteria and viruses
- further enhancement of sensitivity in immunological measurement methods is strongly demanded. It has become like this.
- Patent Document 1 a method of adding a reaction accelerator in a measurement reagent
- Patent Document 2 a method of adding a large amount of inactive protein in a reaction system
- Patent Document 3 a method in which an antigen or antibody is immobilized on an insoluble carrier and blocked with an immunologically inactive protein or the like, and a blocking agent is denatured by heat
- Patent Document 3 a method in which an antigen or antibody is immobilized on an insoluble carrier and blocked with an immunologically inactive protein or the like, and a blocking agent is denatured by heat
- Patent Document 1 has a problem that a nonspecific reaction may be induced.
- Patent Documents 2 and 3 it is common to use serum-derived albumin as a blocking agent for preventing non-specific reactions.
- serum albumin has a problem that the reactivity may vary greatly depending on the lot difference or a sufficient effect may not be obtained.
- an object of the present invention is to provide purified serum albumin having a small lot difference and an immunological measurement method having high reactivity and less non-specific reaction using the purified serum albumin.
- the present invention 1 is serum albumin used in a suspension of a blocking agent and / or an insoluble carrier in an immunological measurement method, and uses a quartz cell having a light path length of 1.0 cm when a 1% aqueous solution is used. Purified serum albumin mainly composed of a fraction having an absorbance measured at a wavelength of 9.0 mAbs or less.
- the present invention 2 is serum albumin used for a suspension of a blocking agent and / or an insoluble carrier in an immunological measurement method, and a fraction whose bilirubin binding amount per mol is 0.9 mmol or less is a main component. Purified serum albumin. The present invention is described in detail below.
- the present inventors have determined that the absorbance measured at a wavelength of 463 nm using a quartz cell with an optical path length of 1.0 cm in a 1% aqueous solution is 9.0 mAbs or less, or 1 mol
- purified serum albumin mainly composed of a fraction having a bilirubin binding amount of 0.9 mmol or less as a main component is used as a suspension of a blocking agent and / or an insoluble carrier, there is almost no lot difference and the reactivity is low.
- the present inventors have found that immunological measurement can be performed with a high non-specific reaction and the present invention has been completed.
- Serum albumin in blood usually adsorbs bilirubin (yellow pigment component), free fatty acids, and the like, and plays a role in transporting them. It is thought that the conventional lot difference of serum albumin may have occurred due to the presence and quantity ratio of these adsorbates.
- the fraction satisfying the requirements of the present invention 1 and 2 is a fraction with very little adsorbate, and is highly reactive by using purified serum albumin containing this as a main component, and immunologically less nonspecific reaction. It is thought that measurement will be realized.
- the purified serum albumin of the present invention 1 contains as a main component a fraction having an absorbance of 9.0 mAbs or less measured at a wavelength of 463 nm using a quartz cell having an optical path length of 1.0 cm when made into a 1% aqueous solution.
- it is a purified serum albumin mainly composed of a fraction having an absorbance of 8.0 mAbs or less measured at a wavelength of 463 nm using a quartz cell having an optical path length of 1.0 cm when a 1% aqueous solution is used.
- Is purified serum albumin whose main component is a fraction having an absorbance of 7.0 mAbs or less measured at a wavelength of 463 nm using a quartz cell having an optical path length of 1.0 cm in a 1% aqueous solution.
- a fraction of serum albumin is a fraction with very little adsorption, such as bilirubin (yellow pigment component, absorption wavelength 438 nm (absorption wavelength of BSA-bound bilirubin is 463 nm)).
- bilirubin yellow pigment component, absorption wavelength 438 nm (absorption wavelength of BSA-bound bilirubin is 463 nm)
- immunological measurement should be performed with little lot difference, high reactivity and little nonspecific reaction. Can do.
- it is preferable to consist only of the said fraction as a main component in the range which does not inhibit the target effect of this invention, it means that another fraction may mix.
- the amount ratio of other fractions allowed to be mixed varies depending on the fraction. 127T. U.
- the ratio at which the amount of change in absorbance at a wavelength of 700 nm can be maintained at 0.06 Abs or more was determined from Table 5 and FIG. For example, in the case of a fraction whose absorbance measured at a wavelength of 463 nm using a quartz cell having an optical path length of 1.0 cm and a 1% aqueous solution exceeds 9.0 mAbs and is 13.5 mAbs or less, It can be mixed up to about 45% by weight.
- T.W. U. Is an abbreviation for TITER UNITS, which is a unit of anti-treponema antibody titer measured by Mediace TPLA (manufactured by Sekisui Medical Co., Ltd.), which is a treponema antibody measurement kit.
- WHO standard products TEE INTERNATIONAL STANDARD for SYPHILITIC HUMAN SERUM [1st international standard preparation], established in 1958
- 1T. U. 2 mIU.
- 10T. U. Those showing the above are considered positive.
- the purified serum albumin of the present invention 2 is mainly composed of a fraction having a bilirubin binding amount per mol of 0.9 mmol or less.
- it is a purified serum albumin whose main component is a fraction whose bilirubin binding amount per mol is 0.8 mmol or less, more preferably a fraction whose bilirubin binding amount per mol is 0.7 mmol or less is a main component.
- Purified serum albumin When the purified serum albumin of the present invention comprising such a fraction of serum albumin as a main component is used in a suspension of a blocking agent and / or an insoluble carrier, there is almost no lot difference, high reactivity, Immunological measurement with less specific reaction can be performed.
- it is preferable to consist only of the said fraction as a main component, in the range which does not inhibit the target effect of this invention, it means that another fraction may mix.
- the amount of binding of bilirubin per mol of serum albumin should be measured, for example, by the azobilirubin method using a bilirubin measuring reagent (Sekisui Medical Co., Ltd., “Autosera BIL-2”, “Autosera D-BIL-2”). Can do.
- the amount ratio of other fractions allowed to be mixed varies depending on the fraction. 127T. U.
- the ratio at which the amount of change in absorbance at a wavelength of 700 nm can be maintained at 0.06 Abs or more was determined from Table 5 and FIG.
- a fraction whose bilirubin binding amount per mol exceeds 0.9 mmol and is 1.5 mmol or less it can be mixed up to about 45% by weight of the whole.
- a fraction having a bilirubin binding amount per mol exceeding 0.9 mmol and not more than 2.0 mmol it can be mixed up to about 15% by weight of the whole.
- a fraction whose bilirubin binding amount per mol exceeds 0.9 mmol and is 2.7 mmol or less it can be mixed up to about 5% by weight of the whole.
- the fraction whose absorbance measured at a wavelength of 463 nm using a quartz cell with an optical path length of 1.0 cm when using the above 1% aqueous solution is 9.0 mAbs or less, or the amount of bilirubin binding per mol is 0.9 mmol or less
- Serum albumin used as the raw material for the purified serum albumin of the present invention is albumin derived from animal serum, and specifically, for example, serum derived from the serum of large mammals such as humans, cows, horses, and sheep is preferable. .
- bovine serum-derived serum albumin is particularly suitable because it is inexpensive and can be obtained in large quantities.
- the serum albumin is roughly purified by a modified corn method, a heat shock method or the like before performing anion exchange chromatography purification.
- the column carrier used for the above-described anion exchange chromatography purification is not particularly limited.
- weak anionic DEAE Separose Fast Flow ANX Separose 4Fast Flow
- strong anionic Q Separose Fast Flow Q Sepharose XL, etc. Is mentioned.
- the preferred lower limit of the particle size of the carrier of the column used for the anion exchange chromatography purification is 10 ⁇ m, and the preferred upper limit is 200 ⁇ m.
- the particle size of the carrier of the column is less than 10 ⁇ m, the back pressure increases, and the flow rate during purification may have to be slowed down.
- the particle size of the column support exceeds 200 ⁇ m, the resolution may be deteriorated.
- a more preferable lower limit of the particle size of the carrier of the column is 45 ⁇ m, and a more preferable upper limit is 165 ⁇ m.
- HiPrep16 / 10DEAE FF HiPrep16 / 10ANX FF (high sub), HiPrep16 / 10Q FF (all manufactured by GE Healthcare) may be used as the column used for the above-described anion exchange chromatography purification.
- a buffer solution (hereinafter also referred to as a binding solution) used when serum albumin is bound to a column carrier in the anion exchange chromatography purification is not particularly limited, and examples thereof include a phosphate buffer, a glycine buffer, and a Tris buffer. Etc.
- the concentration of the buffer solution is usually in the range of 5 to 150 mM.
- the pH of the buffer solution is preferably 4 to 9, and more preferably 5 to 8.
- Examples of the eluate used for the anion exchange chromatography purification include those having a salt concentration of 150 mM or less.
- a phosphate buffer solution, a glycine buffer solution, a Tris buffer solution or the like has a salt concentration of 150 mM or less.
- the salt is not particularly limited, and examples thereof include sodium chloride (NaCl) and potassium chloride (KCl).
- the pH of the eluate is preferably 4 to 9, and more preferably 5 to 8. Usually, however, a solution obtained by adding a salt to the same buffer as the binding solution is used.
- a preferred example of the purification method by the anion exchange chromatography will be described.
- a solution in which serum albumin is added to a binding solution for example, 50 mM Tris-HCl buffer (pH 7.5)
- a binding solution for example, 50 mM Tris-HCl buffer (pH 7.5)
- serum albumin is adsorbed on the anion exchange column while flowing the binding solution.
- serum albumin is eluted by flowing an eluate having a salt concentration of 150 mM or less to obtain purified serum albumin.
- the purified serum albumin obtained by the above anion exchange chromatography purification may be further purified by a dialysis method, a gel filtration method or the like.
- the purified serum albumin of the present invention has very little lot difference.
- immunological measurement with high reactivity and less non-specific reaction can be performed.
- An immunoassay reagent using the purified serum albumin of the present invention is also one aspect of the present invention.
- an immunological measurement method using antigen-antibody reaction an immunological measurement method using the purified serum albumin of the present invention as a suspension of a blocking agent and / or an insoluble carrier is also one aspect of the present invention.
- the reagent for solid phase preparation used for immunological measurement based on the purified serum albumin of the present invention is also one aspect of the present invention.
- the monomer obtained by purifying the purified serum albumin of the present invention by gel filtration is further used to improve the reactivity of the immunological measurement reagent.
- Examples of the substance to be measured that is an object of the immunological measurement method of the present invention include an antigen or an antibody in a biological sample.
- Specific examples include hepatitis (B-type and C-type) -derived antigens or antibodies, HIV antigens or antibodies, syphilis-derived antibodies, cancer markers such as ⁇ -fetoprotein, hormones such as insulin, and tachoids.
- anti-treponema paridum antibody which is an antigen derived from syphilis
- the antigen used in the above anti-treponema / paridum antibody measurement system may be a crushed cell or a purified product.
- combined artificially by the gene recombination technique and what combined 1 or more types may be used.
- the immunological measurement method of the present invention is not particularly limited, but a method using an insoluble carrier in which an antigen or antibody is supported is preferable.
- the insoluble carrier is not particularly limited, and examples thereof include organic polymer powders, microorganisms, blood cells, and cell membrane pieces. Of these, organic polymer powder is preferred.
- the organic polymer powder is not particularly limited, and examples thereof include natural polymer powder and synthetic polymer powder.
- the natural polymer powder is not particularly limited, and examples thereof include insoluble agarose, cellulose, and insoluble dextran.
- the synthetic polymer powder is not particularly limited.
- examples thereof include a polymer and a vinyl acetate-acrylic acid ester copolymer.
- the insoluble carrier those having a sulfonic acid group or a carboxyl group introduced on the surface thereof can also be used.
- the insoluble carrier is particularly preferably latex particles in which a synthetic polymer powder is uniformly suspended.
- the particle size of the latex particles is not particularly limited, but a preferable lower limit is 0.05 ⁇ m and a preferable upper limit is 1.5 ⁇ m.
- a preferable lower limit is 0.05 ⁇ m and a preferable upper limit is 1.5 ⁇ m.
- the particle size of the latex particles is less than 0.05 ⁇ m, the amount of optical change due to aggregation is small, and high sensitivity necessary for measurement may not be obtained.
- the particle size of the latex particles exceeds 1.5 ⁇ m the amount of optical change due to aggregation of the latex particles exceeds the measurable range, and the measurement range may be reduced.
- the more preferable lower limit of the particle size of the latex particles is 0.1 ⁇ m, and the more preferable upper limit is 0.8 ⁇ m.
- the method for supporting the antigen or antibody on the insoluble carrier is not particularly limited, and a conventionally known method for supporting the antigen or antibody by physical or chemical bonding can be used.
- the purified serum albumin of the present invention is used as a blocking agent for the above-described insoluble carrier (latex particles) carrying an antigen or antibody, or the insoluble carrier contains an antigen or antibody. Or is used as a suspension.
- a specimen is added to the latex particle suspension carrying the antigen or antibody thus obtained, and allowed to react for a certain period of time.
- Measure the analyte in the sample by optically measuring or visually observing the degree of aggregation caused by the antigen-antibody reaction between the antigen or antibody supported on the latex particles after the reaction and the analyte in the sample. can do.
- the method of optically measuring the degree of aggregation is not particularly limited, and the increase / decrease of scattered light intensity, absorbed light intensity, transmitted light intensity, etc. can be changed by selecting the size and concentration of the insoluble carrier particles used and setting the reaction time. taking measurement. These methods can be used in combination.
- the wavelength of light used for the above measurement is preferably 300 to 900 nm.
- Examples of the apparatus used in the above optical measurement method include optical instruments that can detect scattered light intensity, transmitted light intensity, absorbance, and the like, and any biochemical automatic analyzer that is generally used can be used. Can be used.
- the method of visually observing the degree of aggregation is usually a method in which a solution containing a specimen and a latex particle suspension is mixed on a determination plate, the mixture is shaken, and then the presence or absence of aggregation is determined. Can be used.
- a method of photographing the aggregation state with a video camera or the like and performing image processing in addition to the visual method.
- the present invention it is possible to provide purified serum albumin having a small lot difference and an immunological measurement method having high reactivity and less non-specific reaction using the purified serum albumin.
- Example 1 Preparation of purified serum albumin
- Commercial bovine serum albumin manufactured by Serological, “Cohn Fraction V”, hereinafter referred to as “unpurified BSA”, Lot83
- a binding solution 50 mM Tris-HCl buffer (pH 7.5)).
- To prepare a 15% crude BSA solution 5 mL of the obtained 15% unpurified BSA solution was added to a commercially available anion exchange column (GE Healthcare, “DEAE Sepharose FF” 480 mL / “XK50”) equilibrated with the binding solution, and the binding solution was 4 mL / min.
- the BSA was adsorbed on the column while flowing through the column.
- an eluate in which a 2M sodium chloride solution was mixed with the binding solution so that the sodium chloride concentration was 80 mM was flowed at a flow rate of 10 mL / min to obtain an albumin fraction eluted with an eluent having a salt concentration of 80 mM.
- a 2M sodium chloride solution is mixed with the binding solution so that the sodium chloride concentration is increased by 2 mM per minute while flowing at a flow rate of 10 mL / min, and after mixing starts from 73.3 to 74.5.
- the albumin fraction eluted in the fraction (salt concentration 69.6-72.0 mM fraction) was obtained.
- the salt concentration of the obtained albumin fraction was the sodium chloride concentration in the albumin solution eluted from the column in consideration of the column volume.
- the obtained fraction solution was dialyzed against 100 mM phosphate buffer (pH 7.4) to adjust to 1%.
- the absorbance at a wavelength of 463 nm of the obtained 1% solution of purified serum albumin was measured using a standard cell (manufactured by GL Sciences, “S10-UV”, optical path length 1.0 cm). .1 mAbs.
- the amount of bilirubin binding per 1 mol of BSA was determined by the azobilirubin method using a bilirubin measuring reagent (manufactured by Sekisui Medical, “Autosera BIL-2”, “Autosera D-BIL-2”).
- the obtained liquid is centrifuged at 13,000 rpm for 10 minutes at 10 ° C., and the obtained precipitate is added to 4 mL of purified BSA solution (1.3 mAbs / 0.1 mmol) to suspend the latex.
- BSA solution 1.3 mAbs / 0.1 mmol
- Example 2 While mixing a 2M sodium chloride solution into the binding solution so that the sodium chloride concentration is increased by 2 mM per minute, the flow rate was 10 mL / min, and 87.7 to 88.9 minutes after the start of mixing (salt concentration of 98.4).
- Example 3 While mixing a 2M sodium chloride solution into the binding solution so that the sodium chloride concentration is increased by 2 mM per minute, the flow rate was 10 mL / min, and 102.1 to 103.3 minutes (salt concentration 127.2) after mixing started.
- a latex solution carrying anti-treponema and paridum antigen was prepared in the same manner as in Example 1 except that a purified BSA (3.3 mAbs / 0.3 mmol) solution eluted in ( ⁇ 129.6 mM fraction) was used as a blocking agent. did.
- Example 1 An anti-treponema / paridum antigen-carrying latex solution was prepared in the same manner as in Example 1 except that the BSA solution was used without purification by anion exchange chromatography (hereinafter referred to as unpurified BSA).
- the absorbance at a wavelength of 463 nm measured in the same manner as in Example 1 was 9.8 mAbs, and the amount of bilirubin binding per 1 mol of BSA was 1.0 mmol.
- anti-treponema / paridum antibody standard solution As anti-treponema / paridum antibody standard solution, 15 ⁇ L of syphilis positive standard serum (Sekisui Medical Co., Ltd., 5 concentration) is collected, and sample dilution solution (containing 1% BSA) is collected. 150 ⁇ L of Lipidure (manufactured by NOF Corporation) 0.2% (w / v)%) was mixed with 100 mM phosphate buffer (pH 7.4) and maintained at 37 ° C. as appropriate.
- Negative sample measurement Absorbance change was obtained in the same manner as (1) except that physiological saline and negative samples 1 to 10 were used as samples, and prepared based on the standard product measurement results of (1) The antibody titer was calculated from the calibration curve. The results are shown in Table 2.
- Example 4 Three kinds of commercially available bovine serum albumins (Lot83, Lot91, Lot77) were prepared. For each of these three lots of bovine serum albumin, an anti-treponema paridum antigen-supporting latex solution was prepared in the same manner as in Example 1.
- Comparative Example 5 An anti-treponema paridum antigen-carrying latex solution was prepared in the same manner as in Comparative Example 1, except that commercially available bovine serum albumin (Lot 83, Lot 91, Lot 77) was used unpurified.
- syphilis positive standard serum (manufactured by Sekisui Medical Co., Ltd., 5 concentration) is collected as an anti-treponema / paridum antibody standard solution, and this is added to a sample diluent (100 mM phosphate buffer (pH 7.4) containing 1% BSA).
- sample diluent 100 mM phosphate buffer (pH 7.4) containing 1% BSA).
- 150 ⁇ L of Lipidure (manufactured by NOF Corporation) with 0.2 (w / v)% added was mixed and kept at 37 ° C. in a timely manner.
- Example 5 The purified BSA solution (1.3 mAbs / 0.1 mmol) obtained in Example 1 was concentrated, added to a commercially available gel filtration column Sephacryl S-200HR (manufactured by GE Healthcare), and 100 mM phosphate buffer as an eluent. The solution (pH 7.4) was subjected to gel filtration purification, and an anti-treponema / paridum antigen-carrying latex solution was prepared in the same manner as in Example 1 except that BSA consisting only of the monomer fraction was used.
- Example 6 Preparation of Anti-Treponema / Paridum Antigen Latex Liquid Using BSA Containing Only Monomer Fraction Purified by Gel Filtration of Purified BSA Solution (1.3 mAbs / 0.1 mmol) Obtained in Example 1 as a Blocking Agent
- the purified BSA (1.3 mAbs / 0.1 mmol) solution obtained in Example 1 was used, an anti-treponema and paridum antigen-carrying latex solution was prepared.
- Example 7 Preparation of Anti-Treponema / Paridum Antigen Latex Liquid Using BSA Containing Only Monomer Fraction Purified by Gel Filtration of Purified BSA Solution (1.3 mAbs / 0.1 mmol) Obtained in Example 1 as a Blocking Agent
- an anti-treponema-paridum antigen-supporting latex solution was prepared.
- syphilis positive standard serum (manufactured by Sekisui Medical Co., Ltd., 5 concentration) is collected as an anti-treponema / paridum antibody standard solution, and this is added to a sample diluent (100 mM phosphate buffer (pH 7.4) containing 1% BSA).
- sample diluent 100 mM phosphate buffer (pH 7.4) containing 1% BSA).
- 150 ⁇ L of Lipidure (manufactured by NOF Corporation) with 0.2 (w / v)% added thereto was mixed and kept at 37 ° C. in a timely manner.
- syphilis positive standard serum (manufactured by Sekisui Medical Co., Ltd., 5 concentration) is collected as an anti-treponema / paridum antibody standard solution, and this is added to a sample diluent (100 mM phosphate buffer (pH 7.4) containing 1% BSA).
- sample diluent 100 mM phosphate buffer (pH 7.4) containing 1% BSA).
- 150 ⁇ L of Lipidure (manufactured by NOF Corporation) with 0.2 (w / v)% added was mixed and kept at 37 ° C. in a timely manner.
- the present invention it is possible to provide purified serum albumin having a small lot difference and an immunological measurement method having high reactivity and less non-specific reaction using the purified serum albumin.
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Abstract
Description
しかし、近年、血液中の癌マーカー、ウイルス等の抗原、細菌やウイルスに対する抗体等の極微量成分を測定するニーズが高まってきており、免疫学的測定方法の更なる高感度化が強く求められるようになってきた。
一方、特許文献2、3に記載された方法では、非特異的な反応を防止するためのブロッキング剤として血清由来のアルブミンを用いることが一般的である。しかしながら、血清アルブミンは、ロット差により反応性が大きく異なったり、充分に効果が得られなかったりすることがあるという問題があった。
本発明2は、免疫学的測定方法においてブロッキング剤及び/又は不溶性担体の懸濁液に用いる血清アルブミンであって、1molあたりのビリルビン結合量が0.9mmol以下である画分を主成分とする精製血清アルブミンである。
以下に本発明を詳述する。
血清アルブミンのこのような画分は、ビリルビン(黄色色素成分、吸収波長438nm(BSA結合型ビリルビンの吸収波長は463nm))等の吸着が極めて少ない画分であり、このような画分を主成分とする本発明の精製血清アルブミンをブロッキング剤及び/又は不溶性担体の懸濁液に用いた場合には、ロット差がほとんどなく、反応性が高く、非特異反応の少ない免疫学的測定を行うことができる。
なお、主成分とするとは、上記画分のみからなることが好ましいものの、本発明の目的効果を阻害しない範囲においては、他の画分が混入してもかまわないことを意味する。
例えば、1%水溶液としたときに光路長が1.0cmの石英セルを用い463nmの波長にて測定した吸光度が9.0mAbsを超えて13.5mAbs以下である画分の場合には、全体の45重量%程度まで混入可能である。
例えば、1%水溶液としたときに光路長が1.0cmの石英セルを用い463nmの波長にて測定した吸光度が9.0mAbsを超えて20.0mAbs以下である画分の場合には、全体の15重量%程度まで混入可能である。
例えば、1%水溶液としたときに光路長が1.0cmの石英セルを用い463nmの波長にて測定した吸光度が9.0mAbsを超えて26.0mAbs以下である画分の場合には、全体の5重量%程度まで混入可能である。
なお、主成分とするとは、上記画分のみからなることが好ましいものの、本発明の目的効果を阻害しない範囲においては、他の画分が混入してもかまわないことを意味する。
例えば、1molあたりのビリルビン結合量が0.9mmolを超えて1.5mmol以下である画分の場合には、全体の45重量%程度まで混入可能である。
例えば、1molあたりのビリルビン結合量が0.9mmolを超えて2.0mmol以下である画分の場合には、全体の15重量%程度まで混入可能である。
例えば、1molあたりのビリルビン結合量が0.9mmolを超えて2.7mmol以下である画分の場合には、全体の5重量%程度まで混入可能である。
また、上記血清アルブミンは、陰イオン交換クロマトグラフィー精製を行う前に、コーン変法やヒートショック法等で粗精製されていることが好ましい。
上記緩衝液の濃度は、通常5~150mMの範囲で用いられる。
上記緩衝液のpHは4~9が好ましく、5~8がより好ましい。
上記塩は特に限定されず、例えば、塩化ナトリウム(NaCl)、塩化カリウム(KCl)等が挙げられる。
上記溶出液のpHは4~9が好ましく、5~8がより好ましい。
ただし、通常は、結合液と同じ緩衝液に塩が加えられたものが用いられる。
まず、血清アルブミンを結合液(例えば、50mMTris-HCl緩衝液(pH7.5))に添加した溶液を調製する。
次いで、該溶液を結合液で平衡化した陰イオン交換カラムに添加し、結合液を流しながら血清アルブミンを陰イオン交換カラムに吸着させる。
次いで、塩濃度が150mM以下である溶出液を流して血清アルブミンを溶出させ、精製血清アルブミンを得る。
本発明の精製血清アルブミンを免疫学的測定方法におけるブロッキング剤又は不溶性担体の懸濁液として用いれば、反応性が高く、非特異反応の少ない免疫学的測定を行うことができる。
本発明の精製血清アルブミンを用いてなる免疫学的測定試薬もまた、本発明の1つである。
抗原抗体反応を利用した免疫学的測定方法において、ブロッキング剤及び/又は不溶性担体の懸濁液として、本発明の精製血清アルブミンを用いる免疫学的測定方法もまた、本発明の1つである。
本発明の精製血清アルブミンを主成分とする免疫学的測定に用いる固相調製用試薬もまた、本発明の1つである。
本発明の精製血清アルブミンをゲルろ過によって精製した単量体は、更に免疫学的測定試薬の反応性を向上するために用いられる。
本発明の免疫学的測定法の対象とする被測定物質は、例えば、生体試料中の抗原又は抗体が挙げられる。具体的には例えば、肝炎(B型、C型)由来抗原又は抗体、HIV抗原又は抗体、梅毒由来抗体、α-フェトプロテイン等の癌マーカー、インシュリン等のホルモン、オータコイド等が挙げられる。
上記抗トレポネーマ・パリダム抗体測定系に用いる抗原は、菌体破砕物であってもよく、精製物であってもよい。また、遺伝子組み換え技術により人工的に合成されたものを1種又はそれ以上組み合わせたものでもよい。
上記不溶性担体は特に限定されず、例えば、有機高分子粉末、微生物、血球、細胞膜片等が挙げられる。なかでも有機高分子粉末が好適である。
上記有機高分子粉末は特に限定されず、例えば、天然高分子粉末、合成高分子粉末等が挙げられる。
上記天然高分子粉末は特に限定されず、例えば、不溶性アガロース、セルロース、不溶性デキストラン等が挙げられる。
上記合成高分子粉末は特に限定されず、例えば、ポリスチレン、スチレン-スルホン酸(塩)共重合体、スチレン-メタクリル酸共重合体、アクリロニトリル-ブタジエン-スチレン共重合体、塩化ビニル-アクリル酸エステル共重合体、酢酸ビニル-アクリル酸エステル共重合体等が挙げられる。
また、上記不溶性担体は、表面にスルホン酸基やカルボキシル基等を導入したものも用いることができる。
上記ラテックス粒子の粒径は特に限定されないが、好ましい下限は0.05μm、好ましい上限は1.5μmである。上記ラテックス粒子の粒径が0.05μm未満であると、凝集による光学的変化量が小さく、測定に必要な高い感度が得られないことがある。上記ラテックス粒子の粒径が1.5μmを超えると、ラテックス粒子の凝集による光学的変化量が測定可能域を超えてしまい、測定範囲が小さくなることがある。上記ラテックス粒子の粒径のより好ましい下限は0.1μm、より好ましい上限は0.8μmである。
上記測定を行う際の光の波長は、300~900nmが好ましい。
(1)精製血清アルブミンの調製
市販の牛血清アルブミン(セロロジカル社製、「Cohn Fraction V」、以下、「未精製BSA」ともいう、Lot83)を結合液(50mM Tris-HCl緩衝液(pH7.5))に添加し、15%未精製BSA溶液を調整した。
得られた15%未精製BSA溶液5mLを、結合液で平衡化した市販の陰イオン交換カラム(GE Healthcare社製、「DEAE SepharoseFF」480mL/「XK50」)に添加し、結合液を4mL/minで流しながらBSAをカラムに吸着させた。次いで、塩化ナトリウム濃度が80mMになるように2Mの塩化ナトリウム溶液を結合液に混和した溶出液を、流速10mL/minで流し、80mMの塩濃度の溶出液で溶出されたアルブミン画分を得た。また、これとは別に、1分間に塩化ナトリウム濃度が2mMずつ増加するように2Mの塩化ナトリウム溶液を結合液に混和しながら、流速10mL/minで流し、混和開始後73.3~74.5分(塩濃度69.6~72.0mM分画)に溶出されたアルブミン画分を得た。なお、得られたアルブミン画分の塩濃度は、カラム容量を考慮し、カラムから溶出されたアルブミン溶液中の塩化ナトリウム濃度を示した。
また、得られた精製血清アルブミンについて、ビリルビン測定試薬(積水メディカル社製、「オートセラBIL-2」、「オートセラD-BIL-2」)を用いたアゾビリルビン法により、BSA1molあたりのビリルビン結合量を測定したところ、0.1mmol、0.5mmolであった。
以下、それぞれを「精製BSA(1.3mAbs/0.1mmol)」、「精製BSA(5.1mAbs/0.5mmol)」ともいう。
100mMリン酸緩衝液(pH7.4)に150μg/mLの蛋白濃度で溶解した抗トレポネーマ・パリダム抗原液400μLを平均粒径0.4μmのポリスチレンラテックス(固形分10(w/v)%、積水化学工業社製)100μLに添加し、4℃にて1時間攪拌した。
次いで、精製BSA(5.1mAbs/0.5mmol)溶液2mLを添加し、1時間攪拌した。得られた液体を10℃にて10分間、13,000rpmで遠心分離し、得られた沈殿物を、精製BSA溶液(1.3mAbs/0.1mmol)4mLに添加し、ラテックスを懸濁させることにより抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
1分間に塩化ナトリウム濃度が2mMずつ増加するように2Mの塩化ナトリウム溶液を結合液に混和しながら、流速10mL/minで流し、混和開始後87.7~88.9分(塩濃度98.4~100.8mM分画)に溶出される精製BSA(0.9mAbs/0.1mmol)溶液をブロッキング剤として用いたこと以外は、実施例1と同様にして抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
1分間に塩化ナトリウム濃度が2mMずつ増加するように2Mの塩化ナトリウム溶液を結合液に混和しながら、流速10mL/minで流し、混和開始後102.1~103.3分(塩濃度127.2~129.6mM分画)に溶出される精製BSA(3.3mAbs/0.3mmol)溶液をブロッキング剤として用いたこと以外は、実施例1と同様にして抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
BSA溶液を陰イオン交換クロマト精製せずに(以下、未精製BSAという)用いたこと以外は、実施例1と同様にして抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
また、未精製BSAの1%溶液について、実施例1と同様にして測定した波長463nmでの吸光度は9.8mAbs、BSA1molあたりのビリルビン結合量は、1.0mmolであった。
1分間に塩化ナトリウム濃度が2mMずつ増加するように2Mの塩化ナトリウム溶液を結合液に混和しながら、流速10mL/minで流し、混和開始後116.5~117.7分(塩濃度156.0~158.4mM分画)に溶出される精製BSA(9.8mAbs/1.0mmol)溶液をブロッキング剤として用いたこと以外は、実施例1と同様にして抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
1分間に塩化ナトリウム濃度が2mMずつ増加するように2Mの塩化ナトリウム溶液を結合液に混和しながら、流速10mL/minで流し、混和開始後130.9~132.1分(塩濃度184.8~187.2mM分画)に溶出される精製BSA(17.9mAbs/1.9mmol)溶液をブロッキング剤として用いたこと以外は、実施例1と同様にして抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
1分間に塩化ナトリウム濃度が2mMずつ増加するように2Mの塩化ナトリウム溶液を結合液に混和しながら、流速10mL/minで流し、混和開始後145.3~146.5分(塩濃度213.6~216.0mM分画)に溶出される精製BSA(24.9mAbs/2.6mmol)溶液をブロッキング剤として用いたこと以外は、実施例1と同様にして抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
実施例1~3、比較例1~4にて調製した抗トレポネーマ・パリダム抗原担持ラテックス液を用いて、下記の方法により評価を行った。
抗トレポネーマ・パリダム抗体標準液として、梅毒陽性標準血清(積水メディカル社製、5濃度)15μLを採取し、これに検体希釈液(BSAを1%含有する100mMリン酸緩衝液(pH7.4)にLipidure(日油社製)を0.2(w/v)%添加したもの)150μLを混和し、37℃で適時保持した。これに、抗トレポネーマ・パリダム抗原担持ラテックス液50μLを添加、撹拌した後、約80秒から300秒までの間の波長700nmでの吸光度の変化量を測定し、吸光度変化量(ΔAbs)とした。
なお、測定は自動分析装置日立7170形を使用した。
結果を表1に示した。
生理食塩水及び陰性検体1~10を検体とした以外は(1)と同様にして吸光度変化量(ΔAbs)を求め、(1)の標準品測定結果に基づいて作成した検量線から抗体価を算出した。結果を表2に示した。
市販の牛血清アルブミンとして3種のロットのもの(Lot83、Lot91、Lot77)を準備した。これらの3種のロットの牛血清アルブミンの各々について実施例1と同様にして抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
市販の牛血清アルブミンとして3種のロットのもの(Lot83、Lot91、Lot77)を未精製のまま用いたこと以外は、比較例1と同様にして抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
実施例4、比較例5にて調製した抗トレポネーマ・パリダム抗原担持ラテックス液を用いて、下記の方法により評価を行った。
なお、測定は自動分析装置日立7170形を使用した。
結果を表3に示した。
実施例1で得られた精製BSA溶液(1.3mAbs/0.1mmol)を濃縮し、市販のゲルろ過カラムSephacryl S-200HR(GE Healthcare社製)に添加し、溶離液として100mMのリン酸緩衝液(pH7.4)を用いてゲルろ過精製し、その単量体画分のみからなるBSAを用いたこと以外は実施例1と同様にして、抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
実施例1で得られた精製BSA溶液(1.3mAbs/0.1mmol)をゲルろ過精製した単量体画分のみからなるBSAをブロッキング剤としてのみ用い、抗トレポネーマ・パリダム抗原担持ラテックス液の調製には実施例1で得られた精製BSA(1.3mAbs/0.1mmol)溶液を用いたこと以外は実施例5と同様にして、抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
実施例1で得られた精製BSA溶液(1.3mAbs/0.1mmol)をゲルろ過精製した単量体画分のみからなるBSAをブロッキング剤としてのみ用い、抗トレポネーマ・パリダム抗原担持ラテックス液の調製には未精製BSA溶液を用いた以外は実施例5と同様にして、抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
実施例5~7にて調製した抗トレポネーマ・パリダム抗原担持ラテックス液を用いて、下記の方法により評価を行った。
なお、測定は自動分析装置日立7170形を使用した。
結果を表4に示した。
ブロッキング剤として表5に記載した比率で混合したBSA溶液を用いた以外は実施例1と同様にして抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
参考例1~10にて調製した抗トレポネーマ・パリダム抗原担持ラテックス液を用いて、下記の方法により評価を行った。
なお、測定は自動分析装置日立7170形を使用した。
結果を表5に示した。
また、横軸を混入BSA比率、縦軸を吸光度変化量として表5の結果をプロットしたグラフを図1に示した。
Claims (8)
- 免疫学的測定方法においてブロッキング剤及び/又は不溶性担体の懸濁液に用いる血清アルブミンであって、1%水溶液としたときに光路長が1.0cmの石英セルを用い463nmの波長にて測定した吸光度が9.0mAbs以下である画分を主成分とすることを特徴とする精製血清アルブミン。
- 1%水溶液としたときに光路長が1.0cmの石英セルを用い463nmの波長にて測定した吸光度が9.0mAbs以下である画分を主成分とする精製血清アルブミンを用いてなることを特徴とする免疫学的測定試薬。
- 抗原抗体反応を利用した免疫学的測定方法において、ブロッキング剤及び/又は不溶性担体の懸濁液に、1%水溶液としたときに光路長が1.0cmの石英セルを用い463nmの波長にて測定した吸光度が9.0mAbs以下である画分を主成分とする精製血清アルブミンを用いることを特徴とする免疫学的測定方法。
- 請求項1記載の精製血清アルブミンを主成分とすることを特徴とする免疫学的測定に用いる固相調製用試薬。
- 免疫学的測定方法においてブロッキング剤及び/又は不溶性担体の懸濁液に用いる血清アルブミンであって、1molあたりのビリルビン結合量が0.9mmol以下である画分を主成分とすることを特徴とする精製血清アルブミン。
- 1molあたりのビリルビン結合量が0.9mmol以下である画分を主成分とする精製血清アルブミンを用いてなることを特徴とする免疫学的測定試薬。
- 抗原抗体反応を利用した免疫学的測定方法において、ブロッキング剤及び/又は不溶性担体の懸濁液に、1molあたりのビリルビン結合量が0.9mmol以下である画分を主成分とする精製血清アルブミンを用いることを特徴とする免疫学的測定方法。
- 請求項5記載の精製血清アルブミンを主成分とすることを特徴とする免疫学的測定に用いる固相調製用試薬。
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US8445213B2 (en) | 2013-05-21 |
EP2261661A1 (en) | 2010-12-15 |
JPWO2009123060A1 (ja) | 2011-07-28 |
JP4544437B2 (ja) | 2010-09-15 |
JP4452324B2 (ja) | 2010-04-21 |
CN101978268A (zh) | 2011-02-16 |
EP2261661A4 (en) | 2011-04-20 |
CN101978268B (zh) | 2014-04-23 |
JP2010048818A (ja) | 2010-03-04 |
US20110027912A1 (en) | 2011-02-03 |
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