WO2008069002A1 - Gold colloid for in vitro diagnostic - Google Patents

Gold colloid for in vitro diagnostic Download PDF

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Publication number
WO2008069002A1
WO2008069002A1 PCT/JP2007/071987 JP2007071987W WO2008069002A1 WO 2008069002 A1 WO2008069002 A1 WO 2008069002A1 JP 2007071987 W JP2007071987 W JP 2007071987W WO 2008069002 A1 WO2008069002 A1 WO 2008069002A1
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Prior art keywords
gold colloid
compound
linker
gold
antibody
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PCT/JP2007/071987
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French (fr)
Japanese (ja)
Inventor
Hirofumi Nakagawa
Original Assignee
Tanaka Kikinzoku Kogyo K. K.
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Publication of WO2008069002A1 publication Critical patent/WO2008069002A1/en

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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
    • G01N33/532—Production of labelled immunochemicals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
    • B22F1/05—Metallic powder characterised by the size or surface area of the particles
    • B22F1/054—Nanosized particles
    • B22F1/0545—Dispersions or suspensions of nanosized particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F2999/00—Aspects linked to processes or compositions used in powder metallurgy

Definitions

  • the present invention relates to a colloidal gold suitable as an in vitro diagnostic agent.
  • a linker is a compound that enables immobilization of an antibody in a form such as a covalent bond.
  • Patent Document 1 discloses a linker composed of non-crosslinked dextran or aminodextran having an SH group.
  • Patent Document 2 discloses a gold colloid having an alkanethiol, an alkanethiol derivative, a dithiol compound, and a trithiol compound as a linker.
  • Patent Document 1 Special Table 2003-536074
  • Patent Document 2 JP-A-6-116602
  • the above in-vitro diagnostic agents are required to improve the accuracy and early detection of the diagnosis.
  • the presence or absence of the target antigen can be confirmed by clear coloration, and detection is possible even when the amount of antigen is small. It is required to be.
  • gold colloids used for in-vitro diagnostic agents are preferably those that react with a small amount of antigen with high color intensity and exhibit sufficient color development.
  • gold colloids with various particle sizes may be required depending on the type of target antigen, etc., so gold colloids that exhibit good color development over a wide range of particle sizes are useful as in vitro diagnostic agents. High availability.
  • the colloidal gold is more suitable as an in vitro diagnostic agent when it satisfies the above-mentioned properties!
  • the gold colloid described in Patent Document 1 and Patent Document 2 described above it is possible to prevent aggregation of the gold colloid and fix the target antibody specifically, but it is particularly suitable as an in vitro diagnostic agent. However, it did not satisfy all of the above characteristics to make a gold colloid.
  • the present invention aims to provide a gold colloid suitable as an in vitro diagnostic agent, which reacts with a small amount of antigen with high color development intensity and exhibits sufficient color development for in vitro diagnosis. And the present invention also provides gold colloids for in vitro diagnostic agents that exhibit good color development over a wide range of particle sizes. Means for solving the problem
  • the present invention is an in vitro diagnostic gold colloid comprising a gold colloid and a linker for immobilizing an antibody by modifying the gold colloid, wherein the linker is represented by chemical formula 1 and / or chemical formula 2
  • the substituents RR and RR are represented by chemical formula 1 and / or chemical formula 2
  • the compound represented by Chemical Formula 1 or Chemical Formula 2 in the present invention has a disulfide group or a thiol group at the terminal of the alkyl group, and has a substituent R or R at the other end of the alkyl group.
  • Disulfide group and thiol group are substituents that act when modifying gold colloid, and substituents R and R are substituents that act when immobilizing an antibody.
  • colloidal gold of the present invention includes two types of substituents R R and / or charcoal.
  • an in vitro diagnostic agent that reacts with a small amount of antigen with high intensity of color development and exhibits good color development can be obtained.
  • Such an effect can be obtained by selecting the substituent R, R and / or the carbon number n, n in the case of the linker of the present invention.
  • the number of antibodies immobilized per gold colloid particle can be adjusted, and the color intensity per gold colloid particle can be used efficiently.
  • Each of the above-mentioned substituents has different reactivity for binding to an antibody, and by using two or more compounds containing substituents having different reactivity, the number of antibodies binding to the gold colloid can be adjusted. Conceivable. Also, with carbon number n, n force 3 ⁇ 4 ⁇ ;
  • Using two or more different compounds can also cause differences in reactivity between colloidal gold and antibodies. It is for using.
  • the linker of the present invention those containing both compounds represented by Chemical Formulas 1 and 2 can be used.
  • the compound represented by Chemical Formula 1 and the compound represented by Chemical Formula 2 are different in the ability to have a disulfide group at one end of the alkyl group and whether to have a thiol group. It becomes a thiol group, and the thiol group becomes a disulfide group by oxidation. Therefore, the compound represented by Chemical Formula 1 and the compound represented by Chemical Formula 2 are highly compatible, so it can be considered that they can be mixed and used.
  • the compound represented by Chemical Formula 1 and the compound represented by Chemical Formula 2 can be used in combination.
  • the linker applied in the present invention is represented by Chemical Formula 1 or Chemical Formula 2. It preferably consists of only one of the compounds. This is because disulfide groups and thiol groups are difficult to adjust to the target reactivity due to differences in reactivity with antibodies. That is, when a compound represented by Chemical Formula 1 and a compound represented by Chemical Formula 2 are used in combination, the substituents R, R
  • the linker is composed of a first compound in which the substituent R or R is a carboxyl group, and the substituent R or R is a carboxyl group.
  • the substituent R or R of the second compound described above is
  • a 1 2 hydroxyl group is preferred.
  • the carboxyl group is known as one of the substituents that are highly bound to the amino group in the protein, and the hydroxyl group is known as a substituent that does not react well with either the amino group or the carboxyl group in the protein. Yes. Therefore, it is considered that the number of antibodies immobilized on the gold colloid can be adjusted more accurately by using a linker containing a carboxyl group having a high reactivity with an antibody and a hydroxyl group having a low reactivity.
  • the first compound having a carboxyl group and a substituent other than the carboxyl group.
  • the second compound is a linker
  • the difference between the number of carbon atoms of the first compound and the number of carbon atoms of the second compound is preferably 2 or more.
  • the difference in the reactivity with the antibody also occurs due to the difference in the number of carbon atoms.
  • the first compound is a compound having carbon number n or n force ⁇ ; 10 and the second compound has carbon number n or
  • a compound having a 1 2 1 n force of 3 to 8 is preferred.
  • colloidal gold is used as an in vitro diagnostic agent
  • the color development intensity can be high, and it can also develop color by reacting with a small amount of antigen.
  • the mixing ratio of both compounds is 1: 1 to It is preferably 0.1: 9.9, more preferably 1: 1 to 1: 9. If it is a ratio of 1: 1 to 0.1: 9.9, it will be a gold colloid showing good color development as an in vitro diagnostic agent, and if it is 1:;! To 1: 9, it will have a higher color intensity. be able to.
  • the particle size of the gold colloid is large, increasing the proportion of the second compound reduces the reactivity with the antibody, so that the number of antibodies immobilized per gold colloid particle does not become excessive.
  • colloidal gold particle size is large, the number of antibodies immobilized per colloidal gold colloid will increase, and if the number of antibodies immobilized immediately is large, when used as an in vitro diagnostic agent, the amount will be higher than that required for diagnosis. Excess antigen may be required.
  • the gold colloid of the present invention preferably has a particle size of 10-10 Onm, more preferably 40-10 Onm.
  • Gold colloid with a particle size of 10 nm to 150 nm can be used as an in-vitro diagnostic agent. If the particle size is 40 to 100 nm, it can be expressed with sufficient power S.
  • the gold colloid of the present invention described above can be used for diagnosis by an immunochromatographic method.
  • a moving layer for developing a sample, a colloidal gold provided on one end of the moving layer, to which an antibody corresponding to an antigen is immobilized, and the other moving layer is provided.
  • a force S for using an immunochromatographic diagnostic kit comprising a portion for determining the presence or absence of an antigen contained in the end, and the gold colloid for in vitro diagnostic agents of the present invention can be used as this gold colloid.
  • FIG. 1 is a graph of color intensity of the first embodiment.
  • FIG. 2 is a graph of color intensity of the second embodiment.
  • FIG. 3 is a graph of color intensity of the third embodiment.
  • FIG. 4 is a graph of color intensity of the fourth embodiment.
  • the antibody was fixed to prepare a gold colloid for in-vitro diagnostics (hereinafter referred to as antibody-fixed gold colloid). Then, after preparing a test strip for use in immunochromatography, immunochromatography was performed using the antibody-immobilized gold colloid and the antigen, and the intensity of color developed by the gold colloid was measured.
  • Gold colloid with an average particle size of 40 nm was formed by the following method. Chloroauric acid tetrahydrate 0.17 g and trisodium citrate dihydrate 0 ⁇ 49 g were dissolved in 25 ml and 100 ml of ultrapure water, respectively, to prepare a chloroauric acid solution and a citrate solution. . Next, 6 ml of chloroauric acid solution and 200 ml of pure water were put into a 500 ml triloflasco and heated to reflux for 30 minutes. After the liquid temperature was stabilized, 50 ml of citrate solution was mixed and heated to reflux for 15 minutes. Thereafter, heating was stopped and the mixture was allowed to cool at room temperature to form a nuclear colloid.
  • a gold colloid having an average particle diameter of 80 nm was formed using the gold colloid having an average particle diameter of 40 nm obtained as described above.
  • 33.3 ml of colloidal gold having an average particle size of 40 nm was placed in a 500 ml tri-lasco and stirred in the thermostatic layer until the liquid temperature reached 30 ° C.
  • 118 ml of chloroauric acid solution in which 0.039 g of chloroauric acid tetrahydrate is dissolved and 120 ml of L ascorbic acid solution in which 0.043 g of sodium lascorbate is dissolved are added dropwise simultaneously. , 1 The reaction was allowed to stir for an hour.
  • the colloidal gold of lOOnm is composed of 247ml of chloroauric acid solution in which 0.079g of chloroauric acid tetrahydrate is dissolved in 33.4ml of gold colloid with an average particle size of 40nm, and sodium L-ascorbate.
  • the gold particle colloid formed by the above method with a particle size of 40, 80, lOOnm,
  • Each of the first to fourth embodiments includes the particle size of the gold colloid, the types of the first compound and the second compound used as the linker, and the first compound and the second compound.
  • the molar ratio is shown in Table 1.
  • a colloidal gold having a particle size of 80 nm is modified with a linker containing only a compound of formula 1 (having a disulfide group) and only a compound of formula 2 (having a thiol group).
  • a linker containing 1 was modified.
  • the linker including the first compound having a carbon number n of 3, 5, 7, 10 is modified.
  • the particle size of the gold colloid is 40 nm, 80 nm, or lOOnm
  • the molar ratio of the first compound to the second compound is 10: 0, 5: 5, 1: 9.
  • the linker I was modified.
  • a linker containing only the first compound is modified with a colloidal gold, and the first compound having a carbon number n force S 10 and the carbon number n is 3, 5, 7, A linker containing 10 first compound at a molar ratio of 1: 9 was used.
  • Gold colloid 1st compound (A) 2nd compound (B) Molar ratio Granules (A: B) 1st real bowl HOOC (CH) 10 -S OH (CH) 2 -S
  • a specific method for modifying the linker will be described with reference to a gold colloid having a molar ratio of 5: 5 in the third embodiment as an example.
  • To 100 ml of gold colloid obtained by the above method add 5 ml of methanol solution (concentration 10 M) of the first compound, which is the linker, and 5 ml of methanol solution (concentration 10 M) of the second compound, at room temperature. The reaction was performed for 24 hours. Thereafter, dialysis was performed 3 times for 5 hours using 1 L of 10 mM borate buffer (pH 8.1) to remove the unreacted solution to obtain a colloidal gold modified with a linker.
  • the linker was modified by the same method as above.
  • the antibody was immobilized on each gold colloid having a different particle diameter and linker.
  • Antibodies include chorionic gonad-stimulating hormone used to diagnose the establishment of pregnancy Antibody was used.
  • the antibody (anti-human monoclonal antibody clone5008, manufactured by Biochemica) 100 1 was prepared and reacted at room temperature for 3 hours.
  • 10 mM borate buffer solution ( ⁇ 8 ⁇ 1) was used as a buffer solution for force lupoxylimide hydrochloride and chorionic gonadotropin antibody.
  • 1% polyethylene glycolol 20000 (50 mM potassium phosphate buffer (pH 7.5) was used) was 50-1 and 10% urine serum albumin (hereinafter referred to as BSA): 50 mM phosphorus Potassium acid buffer solution (pH 9.0) was used.) 100 1 was added, and the mixture was further reacted for 10 minutes. Thereafter, the mixture was centrifuged at 4 ° C for 15 minutes, and about 90% of the supernatant was removed, and the precipitated antibody-fixed gold colloid was collected. The collected precipitate was ultrasonically dispersed and then stored in a storage solution (1.0% BSA, 0.05% polyethylene glycol 20000, 0.1% NaN,
  • an antibody-immobilized gold colloid was prepared for a gold colloid in which the linker was not modified. 50mM KH PO using gold colloid with average particle size of 40nm, 80nm, lOOnm
  • Test strips were made for immunochromatographic diagnosis.
  • a test line for determining the presence / absence of an antigen on a membrane serving as a moving layer and a control line serving as a guideline for whether or not normal color was developed on a colloidal gold membrane were prepared.
  • the test line is coated with an antibody that captures only the antibody-immobilized gold colloid that forms a complex with the antigen, and the control line is an antibody that captures the antibody-immobilized gold colloid that does not contain the antigen.
  • the body was applied.
  • the test line was prepared by preparing a solution (Table 2) using an anti-ha S antibody as an antibody, and using this solution 0.7 5 1 by a quantitative dispensing device (Biojet Quanti 3000, manufactured by BioDot). It was prepared by applying to a membrane under the conditions of 50 mm / second, 20.83 nL / dot, and 0.28 mm pitch. The membrane was made of nitrocellulose, 25 mm wide and 30 cm long.
  • a control line was prepared on a membrane by preparing a solution (Table 3) using an anti-mouse antibody as an antibody, under the same coating conditions as the test line.
  • the membrane on which the test line and the control line were formed was dried in an incubator at 42 ° C for 1 hour, and further dried at room temperature for 2 hours.
  • the membrane was then immersed in a blocking solution to prevent the antibody-immobilized gold colloid from adsorbing to the nitrocellulose membrane.
  • the blocking solution was 0.5% casein (50 mM borate buffer (pH 8.5)), the membrane was soaked first, and then the whole was immersed and allowed to stand at room temperature for 30 minutes. .
  • the membrane was immersed in 0.01% sodium dodecyl sulfate (5 mM phosphate buffer (pH 7.5)) as a washing solution as described above. Left to stand.
  • the membrane was taken out from the cleaning solution and allowed to stand for 24 hours, and then an absorbent pad was attached and cut to a width of 4 mm.
  • Figures 1 to 4 show the results of calculating the ratio of the colloidal intensity of gold colloid modified with the linker of the first to fourth embodiments to the intensity of colloidal gold without modification of the linker. .
  • the ratio of the color intensity to the gold colloid without modification of the linker is 100% or more regardless of which gold colloid having a particle size of 40, 80, or lOOnm is used, indicating good color development.
  • the molar ratio of the ratio of the first compound to the second compound is 5: 5 (ie 1: 1) for a particle size of 40 nm, and 1: 9 for a particle size of 80 nm and lOOnm. It was found that the color intensity of the colloidal gold was higher.
  • the gold colloid when only the first compound is used has a color development intensity ratio of about 100% with respect to the gold colloid not modified with the linker, and the color development intensity is high.
  • the force that was difficult to develop The relationship was that the color development intensity increased as the number of carbon atoms n of the linker increased, and it was found that the color development intensity was easy to adjust.
  • the gold colloid of the present invention is a gold colloid suitable as an in vitro diagnostic agent. Further, even a trace amount of antigen with high color development intensity can be detected, and the color developability is good over a wide range of particle sizes.

Abstract

It is intended to provide a gold colloid for an in vitro diagnostic which is a gold colloid suitable as an in vitro diagnostic, shows a high color development intensity and reacts even with a small amount of an antigen to thereby show sufficient color development. It is also intended to provide a gold colloid for an in vitro diagnostic which shows favorable color development over a broad scope of particle size. Namely, a gold colloid for an in vitro diagnostic which comprises a gold colloid and a linker for modifying the gold colloid to thereby immobilize an antibody, wherein the above-described linker comprises compounds represented by Chemical formula 1 and/or Chemical formula 2, namely, two or more kinds of compounds differing from each other in the substituents R1 and R2 and/or the carbon atom numbers n1 and n2. The gold colloid as described above is suitable as an in vitro diagnostic, shows a high color development intensity, enables the detection of an antigen even in a small amount and shows favorable color development over a broad scope of particle size.

Description

明 細 書  Specification
体外診断薬用金コロイド  Gold colloid for in vitro diagnostics
技術分野  Technical field
[0001] 本発明は、体外診断薬として好適な金コロイドに関する。  [0001] The present invention relates to a colloidal gold suitable as an in vitro diagnostic agent.
背景技術  Background art
[0002] ィムノクロマト法や生体物質の染色等による体外診断において、金コロイドを用いた 診断方法が知られている。金コロイドを用いた場合、表面プラズモン共鳴により赤色 の強い発色が観察可能となり、視覚による簡易的な診断が行える。また、金コロイドの 発色は、時間経過による退色が少なぐ生体物質等を固定した場合にも観察可能で あるため、体外診断薬として好適である。  [0002] Diagnostic methods using colloidal gold are known for in vitro diagnosis by immunochromatography or staining of biological materials. When colloidal gold is used, strong red coloration can be observed by surface plasmon resonance, and simple visual diagnosis can be performed. Further, the color development of colloidal gold can be observed even when a biological substance or the like that is less likely to fade over time is fixed, and thus is suitable as an in vitro diagnostic agent.
[0003] 体外診断薬における金コロイドの具体的な使用方法としては、ィムノクロマト法等に おいて、金コロイドに抗体を固定した標識粒子を用いる方法が知られている。ィムノク 口マト法における使用例としては、抗原が存在する場合、前記標識粒子に抗原が結 合した複合体を形成させて、移動層上を展開し、対応する抗体の固定された判定部 において複合体を捕捉する方法が知られている。この方法では、判定部において発 色が見られるかどうかにより、抗原の有無を確認することができる。  [0003] As a specific method of using colloidal gold in an in vitro diagnostic agent, a method using labeled particles in which an antibody is immobilized on colloidal gold is known in the immunochromatography method or the like. As an example of use in the Imunoguchi-Mato method, when an antigen is present, a complex in which the antigen is bound to the labeled particles is formed, developed on the moving bed, and then combined in the determination unit to which the corresponding antibody is immobilized. Methods for capturing the body are known. In this method, the presence or absence of an antigen can be confirmed based on whether or not color is seen in the determination unit.
[0004] 前記した標識粒子作成のため、金コロイドに抗体を固定する方法としては、溶媒中 に金コロイドと抗体とを分散させ、物理吸着により固定する方法が知られている。しか し、この方法は、金コロイド粒子同士の凝集を生じやすいものであった。また、金コロ イドに抗体以外のタンパク質が固定されることもあった。  [0004] In order to prepare labeled particles as described above, as a method for immobilizing an antibody on a gold colloid, a method is known in which a gold colloid and an antibody are dispersed in a solvent and immobilized by physical adsorption. However, this method tends to cause aggregation of colloidal gold particles. In addition, proteins other than antibodies may be immobilized on the gold colloid.
[0005] そこで、金コロイド粒子同士の凝集を防ぎ、金コロイドに目的の抗体を選択的に固 定するため、金コロイドをリンカ一で修飾する方法が知られている。リンカ一とは、共 有結合等の形式で抗体の固定化を可能とする化合物であり、例えば、特許文献 1に は、 SH基を有する非架橋デキストラン又はアミノデキストランからなるリンカ一が示さ れている。また、特許文献 2には、アルカンチオール、アルカンチオール誘導体、ジ チオール化合物及びトリチオール化合物をリンカ一とする金コロイドが示されている。 このリンカ一を用いることにより、抗体の特異的吸着を促進することができると共に、 金コロイド同士の凝集も防ぐことができる。 [0005] Therefore, in order to prevent aggregation of colloidal gold particles and selectively fix the target antibody to the colloidal gold, a method of modifying the colloidal gold with a linker is known. A linker is a compound that enables immobilization of an antibody in a form such as a covalent bond. For example, Patent Document 1 discloses a linker composed of non-crosslinked dextran or aminodextran having an SH group. Yes. Patent Document 2 discloses a gold colloid having an alkanethiol, an alkanethiol derivative, a dithiol compound, and a trithiol compound as a linker. By using this linker, the specific adsorption of the antibody can be promoted, Aggregation of gold colloids can also be prevented.
特許文献 1 :特表 2003— 536074号公報  Patent Document 1: Special Table 2003-536074
特許文献 2：特開平 6— 116602号公報  Patent Document 2: JP-A-6-116602
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0006] 上述した体外診断薬では、診断の精度向上や早期発見が望まれており、明確な発 色により目的とする抗原等の有無を確認できること、抗原等が微量の場合にも検出可 能であること等が求められている。従って、体外診断薬に用いる金コロイドとしては、 発色の強度が大きぐ微量の抗原とも反応して充分な発色を示すものが好適である。 また、体外診断においては、 目的の抗原等の種類によって、様々な粒径の金コロイド を必要とする場合があるため、広範な粒径において良好な発色を示す金コロイドは、 体外診断薬としての利用性が高い。  [0006] The above in-vitro diagnostic agents are required to improve the accuracy and early detection of the diagnosis. The presence or absence of the target antigen can be confirmed by clear coloration, and detection is possible even when the amount of antigen is small. It is required to be. Accordingly, gold colloids used for in-vitro diagnostic agents are preferably those that react with a small amount of antigen with high color intensity and exhibit sufficient color development. In addition, in in vitro diagnosis, gold colloids with various particle sizes may be required depending on the type of target antigen, etc., so gold colloids that exhibit good color development over a wide range of particle sizes are useful as in vitro diagnostic agents. High availability.
[0007] 従って、金コロイドは、上記した特性を満たして!/、る場合に、体外診断薬として、より 好適なものとなる。一方、前記した特許文献 1や特許文献 2に記載の金コロイドによれ ば、金コロイドの凝集を防ぎ、 目的とする抗体を特異的に固定することは可能となるが 、体外診断薬として特に好適な金コロイドとするための上記特性を、全て満たしてい るものではなかった。  [0007] Accordingly, the colloidal gold is more suitable as an in vitro diagnostic agent when it satisfies the above-mentioned properties! On the other hand, according to the gold colloid described in Patent Document 1 and Patent Document 2 described above, it is possible to prevent aggregation of the gold colloid and fix the target antibody specifically, but it is particularly suitable as an in vitro diagnostic agent. However, it did not satisfy all of the above characteristics to make a gold colloid.
[0008] そこで、本発明は、体外診断薬として好適な金コロイドであって、発色の強度が大き ぐ微量の抗原とも反応して充分な発色を示す体外診断用の金コロイドの提供を目 的とする。また、本発明は、広範な粒径において、良好な発色を示す体外診断薬用 の金コロイドを提供する。 課題を解決するための手段  [0008] Therefore, the present invention aims to provide a gold colloid suitable as an in vitro diagnostic agent, which reacts with a small amount of antigen with high color development intensity and exhibits sufficient color development for in vitro diagnosis. And The present invention also provides gold colloids for in vitro diagnostic agents that exhibit good color development over a wide range of particle sizes. Means for solving the problem
[0009] 本発明者等は、鋭意検討を行い、上記課題を解決可能とする化合物を含むリンカ 一を見出し、本発明に想到した。すなわち、本発明は、金コロイドと、前記金コロイド を修飾し抗体を固定するためのリンカ一と、を含む体外診断薬用金コロイドであって 、前記リンカ一は、化 1及び/又は化 2で示される化合物からなり、置換基 R R及 [0009] The inventors of the present invention have intensively studied, found a linker containing a compound that can solve the above problems, and have come up with the present invention. That is, the present invention is an in vitro diagnostic gold colloid comprising a gold colloid and a linker for immobilizing an antibody by modifying the gold colloid, wherein the linker is represented by chemical formula 1 and / or chemical formula 2 The substituents RR and
1、 2 び/又は炭素数 n、 nの異なる 2種以上の化合物からなることを特徴とする体外診  In-vitro examination characterized by consisting of two or more compounds with 1, 2 and / or carbon number n, n different
1 2  1 2
断薬用金コロイドに関する。 [化 1]
Figure imgf000005_0001
Figure imgf000005_0002
It relates to a colloidal gold colloid. [Chemical 1]
Figure imgf000005_0001
Figure imgf000005_0002
( =2〜： LO,
Figure imgf000005_0003
OH, CHO, NHa) (= 2 ~: LO,
Figure imgf000005_0003
(OH, CHO, NHa)
[化 2]
Figure imgf000005_0004
[Chemical 2]
Figure imgf000005_0004
(n2=2〜： 10、 R2=COOH, OH, CHO, NH2) (n2 = 2 ～: 10, R 2 = COOH, OH, CHO, NH2)
[0010] 本発明における、化 1又は化 2で示される化合物は、アルキル基の末端にジスルフ イド基又はチオール基を有し、アルキル基の他の端部に置換基 R又は Rを有する。 [0010] The compound represented by Chemical Formula 1 or Chemical Formula 2 in the present invention has a disulfide group or a thiol group at the terminal of the alkyl group, and has a substituent R or R at the other end of the alkyl group.
1 2 ジスルフイド基とチオール基は、金コロイドを修飾する際に作用する置換基であり、置 換基 R、 Rは、抗体を固定する際に作用する置換基である。  1 2 Disulfide group and thiol group are substituents that act when modifying gold colloid, and substituents R and R are substituents that act when immobilizing an antibody.
1 2  1 2
[0011] また、本発明の金コロイドは、置換基 R R及び/又は炭 る 2種  [0011] Further, the colloidal gold of the present invention includes two types of substituents R R and / or charcoal.
1、 素数 n 1, prime number n
2 1、 nの異な 2 1, n different
2  2
以上の化合物からなるリンカ一を含むことにより、発色の強度が大きぐ微量の抗原と も反応して良好な発色を示す体外診断薬とすることができる。このような効果が得ら れるのは、本発明のリンカ一であれば、置換基 R、 R及び/又は炭素数 n、 nを選  By including a linker composed of the above compounds, an in vitro diagnostic agent that reacts with a small amount of antigen with high intensity of color development and exhibits good color development can be obtained. Such an effect can be obtained by selecting the substituent R, R and / or the carbon number n, n in the case of the linker of the present invention.
1 2 1 2 択することにより、金コロイド 1粒子当たりに固定される抗体数を調整でき、金コロイド 1 粒子当たりの発色強度を効率的に利用できるものと考えられる。  By selecting 1 2 1 2, the number of antibodies immobilized per gold colloid particle can be adjusted, and the color intensity per gold colloid particle can be used efficiently.
[0012] 尚、本発明の化 1、化 2で示される化合物は、置換基 R、 R 1 カルボキシル基、ヒ  [0012] The compounds represented by the chemical formulas 1 and 2 of the present invention have the substituent R, R 1 carboxyl group,
1 2  1 2
ドロキシノレ基、ァノレデヒド基、ァミノ基のいずれかであり、炭素数 n、 n力 ¾〜； 10の範  A droxynole group, an aldehyde group, or an amino group, having n carbon atoms, n forces ¾ to 10
1 2  1 2
囲内のものである。上記した置換基は、それぞれ抗体と結合する反応性が異なるもの であり、これら反応性の異なる置換基を含む化合物を 2種以上用いることで、金コロイ ドに結合する抗体数を調整できるものと考えられる。また、炭素数 n、 n力 ¾〜； 10で  It is the one in the range. Each of the above-mentioned substituents has different reactivity for binding to an antibody, and by using two or more compounds containing substituents having different reactivity, the number of antibodies binding to the gold colloid can be adjusted. Conceivable. Also, with carbon number n, n force ¾ ~;
1 2  1 2
異なる 2種以上の化合物を用いることも、同様に、金コロイドと抗体との反応性の違い を利用するためである。 Using two or more different compounds can also cause differences in reactivity between colloidal gold and antibodies. It is for using.
[0013] また、本発明のリンカ一としては、化 1、化 2で示される化合物を両方含むものを利 用できる。化 1で示される化合物と、化 2で示される化合物とは、アルキル基の一端に ジスルフイド基を有する力、、チオール基を有するかという相違点を有している力 ジス ルフイド基は還元によって容易にチオール基となり、チオール基は酸化によってジス ルフイド基となる関係にある。従って、化 1で示される化合物と、化 2で示される化合物 とは、相溶性が高いため、混合して用いること力 Sできるものと考えられる。  [0013] Further, as the linker of the present invention, those containing both compounds represented by Chemical Formulas 1 and 2 can be used. The compound represented by Chemical Formula 1 and the compound represented by Chemical Formula 2 are different in the ability to have a disulfide group at one end of the alkyl group and whether to have a thiol group. It becomes a thiol group, and the thiol group becomes a disulfide group by oxidation. Therefore, the compound represented by Chemical Formula 1 and the compound represented by Chemical Formula 2 are highly compatible, so it can be considered that they can be mixed and used.
[0014] 上記したように、化 1で示される化合物と化 2で示される化合物とは、混合して用い ることもできる力 本発明で適用するリンカ一は、化 1又は化 2で示される化合物のい ずれかのみからなることが好ましい。ジスルフイド基と、チオール基とは、抗体との反 応性に違いがあるため、 目標とする反応性に調整し難くなるためである。すなわち、 化 1で示される化合物と、化 2で示される化合物とを混合して用いると、置換基 R、 R  [0014] As described above, the compound represented by Chemical Formula 1 and the compound represented by Chemical Formula 2 can be used in combination. The linker applied in the present invention is represented by Chemical Formula 1 or Chemical Formula 2. It preferably consists of only one of the compounds. This is because disulfide groups and thiol groups are difficult to adjust to the target reactivity due to differences in reactivity with antibodies. That is, when a compound represented by Chemical Formula 1 and a compound represented by Chemical Formula 2 are used in combination, the substituents R, R
1 2 及び/又は炭素数 n、 nの異なる 2種以上の化合物を含むことにより抗体との反応  1 2 and / or reaction with antibodies by containing two or more compounds with different n and n carbon numbers
1 2  1 2
性を調整した場合であっても、これとは別にジスルフイド基とチオール基との反応性 の相違による影響を考慮しなければならなくなり、 目標とする反応性に調整しに《な ると考えられる。  Even if the reactivity is adjusted, it is necessary to consider the influence of the difference in reactivity between the disulfide group and the thiol group separately, and it is considered that it will be adjusted to the target reactivity .
[0015] 化 1又は化 2で示される化合物のいずれかのみからなる場合、リンカ一は、置換基 R 又は Rがカルボキシル基である第 1の化合物と、置換基 R又は Rがカルボキシル [0015] In the case of consisting of only one of the compounds represented by Chemical Formula 1 or Chemical Formula 2, the linker is composed of a first compound in which the substituent R or R is a carboxyl group, and the substituent R or R is a carboxyl group.
1 2 1 2 1 2 1 2
基以外の置換基である第 2の化合物とを含むことが好ましレ、。抗体との反応性が異な る置換基を含む化合物を用レ、ることにより、金コロイド 1粒子当たりに固定される抗体 の数を調整できると考えられる。また、上記した第 2の化合物の置換基 R又は Rは、  Preferably comprising a second compound that is a substituent other than a group. It is considered that the number of antibodies immobilized per particle of colloidal gold can be adjusted by using a compound containing a substituent having a different reactivity with the antibody. In addition, the substituent R or R of the second compound described above is
1 2 ヒドロキシル基であることが好ましい。カルボキシル基は、タンパク質中のアミノ基との 結合性が高い置換基の 1つとして知られており、ヒドロキシル基は、タンパク質中のァ ミノ基ともカルボキシル基とも、あまり反応しない置換基として知られている。このため 、抗体との反応性が高いカルボキシル基と、反応性の低いヒドロキシル基を含むリン カーを用いれば、金コロイドに固定される抗体の数を、より正確に調整できると考えら れる。  A 1 2 hydroxyl group is preferred. The carboxyl group is known as one of the substituents that are highly bound to the amino group in the protein, and the hydroxyl group is known as a substituent that does not react well with either the amino group or the carboxyl group in the protein. Yes. Therefore, it is considered that the number of antibodies immobilized on the gold colloid can be adjusted more accurately by using a linker containing a carboxyl group having a high reactivity with an antibody and a hydroxyl group having a low reactivity.
[0016] また、カルボキシル基を有する第 1の化合物と、カルボキシル基以外の置換基を有 する第 2の化合物とをリンカ一とする場合、第 1の化合物の炭素数と第 2の化合物の 炭素数との差が、 2以上であることが好ましい。炭素数の差によっても、抗体との反応 性に相違が生じることから、置換基の種類による抗体との反応性調整効果と併用する ことで、金コロイドに固定される抗体の数を、より正確に調整できる。さらに、第 1の化 合物は、炭素数 n又は n力 〜； 10の化合物であり、第 2の化合物は、炭素数 n又は [0016] Further, the first compound having a carboxyl group and a substituent other than the carboxyl group. When the second compound is a linker, the difference between the number of carbon atoms of the first compound and the number of carbon atoms of the second compound is preferably 2 or more. The difference in the reactivity with the antibody also occurs due to the difference in the number of carbon atoms. Can be adjusted. Furthermore, the first compound is a compound having carbon number n or n force ˜; 10 and the second compound has carbon number n or
1 2 1 n力 ¾〜8の化合物であることが好ましい。金コロイドを体外診断薬として用いた場合 A compound having a 1 2 1 n force of 3 to 8 is preferred. When colloidal gold is used as an in vitro diagnostic agent
2 2
に、発色強度の大きなものとし、微量の抗原とも反応して発色を示すものとすることが できる。  In addition, the color development intensity can be high, and it can also develop color by reacting with a small amount of antigen.
[0017] 更に、カルボキシル基を有する第 1の化合物と、カルボキシル基以外の置換基を有 する第 2の化合物とをリンカ一とする場合、両化合物の混合比率は、モル比で 1： 1〜 0. 1 : 9. 9とするのが好ましく、 1 : 1〜； 1 : 9がさらに好ましい。 1 : 1〜0. 1 : 9. 9の割合 であれば、体外診断薬として良好な発色を示す金コロイドとなり、 1：；!〜 1： 9であれば 、より発色強度の大きいものとすることができる。特に、金コロイドの粒径が大きい場合 には、第 2の化合物の割合を多くすることで、抗体との反応性を低くして、金コロイド 1 粒子当たりに固定される抗体数が過剰とならないように調整することが好ましいと考え られる。金コロイドの粒径が大きいと、金コロイド 1粒子当たりに固定される抗体数が多 くなりやすぐ固定される抗体数が多いと、体外診断薬として用いる場合、診断に必 要な量よりも過剰の抗原が必要になると考えられる。  [0017] Furthermore, when the first compound having a carboxyl group and the second compound having a substituent other than a carboxyl group are used as a linker, the mixing ratio of both compounds is 1: 1 to It is preferably 0.1: 9.9, more preferably 1: 1 to 1: 9. If it is a ratio of 1: 1 to 0.1: 9.9, it will be a gold colloid showing good color development as an in vitro diagnostic agent, and if it is 1:;! To 1: 9, it will have a higher color intensity. be able to. In particular, when the particle size of the gold colloid is large, increasing the proportion of the second compound reduces the reactivity with the antibody, so that the number of antibodies immobilized per gold colloid particle does not become excessive. It is considered preferable to make such adjustments. If the colloidal gold particle size is large, the number of antibodies immobilized per colloidal gold colloid will increase, and if the number of antibodies immobilized immediately is large, when used as an in vitro diagnostic agent, the amount will be higher than that required for diagnosis. Excess antigen may be required.
[0018] 尚、本発明の金コロイドは、粒径が 10nm〜150nmであることが好ましぐ 40—10 Onmであることがさらに好ましい。粒径が 10nm〜150nmの金コロイドは、体外診断 薬として利用可能であり、粒径 40〜100nmであれば、充分な発光強度を示すものと すること力 Sでさる。  [0018] The gold colloid of the present invention preferably has a particle size of 10-10 Onm, more preferably 40-10 Onm. Gold colloid with a particle size of 10 nm to 150 nm can be used as an in-vitro diagnostic agent. If the particle size is 40 to 100 nm, it can be expressed with sufficient power S.
[0019] 以上で説明した本発明の金コロイドは、ィムノクロマトグラフ法による診断に使用で きる。具体的には、ィムノクロマトグラフ法では、試料を展開するための移動層と、前 記移動層の一端に設けられ、抗原に対応する抗体が固定された金コロイドと、前記 移動層の他方の端部に含まれる抗原の有無を判定するための部分と、を備えるィム ノクロマトグラフィー用診断キットが用いられる力 S、この金コロイドとして本発明の体外 診断薬用金コロイドを用いることができる。 図面の簡単な説明 [0019] The gold colloid of the present invention described above can be used for diagnosis by an immunochromatographic method. Specifically, in the immunochromatography method, a moving layer for developing a sample, a colloidal gold provided on one end of the moving layer, to which an antibody corresponding to an antigen is immobilized, and the other moving layer is provided. A force S for using an immunochromatographic diagnostic kit comprising a portion for determining the presence or absence of an antigen contained in the end, and the gold colloid for in vitro diagnostic agents of the present invention can be used as this gold colloid. Brief Description of Drawings
[0020] [図 1]第 1実施形態の発色強度のグラフ。  [0020] FIG. 1 is a graph of color intensity of the first embodiment.
[図 2]第 2実施形態の発色強度のグラフ。  FIG. 2 is a graph of color intensity of the second embodiment.
[図 3]第 3実施形態の発色強度のグラフ。  FIG. 3 is a graph of color intensity of the third embodiment.
[図 4]第 4実施形態の発色強度のグラフ。  FIG. 4 is a graph of color intensity of the fourth embodiment.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0021] 以下、本発明における最良の実施形態について説明する。 Hereinafter, the best embodiment of the present invention will be described.
[0022] 金コロイドを形成させて、リンカ一を修飾した後、抗体を固定して体外診断薬用の金 コロイド（以下、抗体固定金コロイドという。）を作成した。そして、ィムノクロマトグラフィ 一に用いるテストストリップを作成した後、抗体固定金コロイドと、抗原とを用いて、ィ ムノクロマトグラフィーを行い、金コロイドによる発色の強度を測定した。  [0022] After the colloidal gold was formed and the linker was modified, the antibody was fixed to prepare a gold colloid for in-vitro diagnostics (hereinafter referred to as antibody-fixed gold colloid). Then, after preparing a test strip for use in immunochromatography, immunochromatography was performed using the antibody-immobilized gold colloid and the antigen, and the intensity of color developed by the gold colloid was measured.
[0023] [金コロイドの形成]  [0023] [Gold colloid formation]
以下の方法により、平均粒径 40nmの金コロイドを形成させた。塩化金酸四水和物 0. 17gと、クェン酸三ナトリウム二水和物 0· 49gとを、それぞれ超純水 25mlと 100m 1に溶解させて、塩化金酸溶液とクェン酸溶液を調製した。次に、 500mlの三ロフラ スコ内に、塩化金酸溶液 6mlと純水 200mlとを投入して、 30分間加熱還流させた。 液温が安定した後、クェン酸溶液 50mlを混合して、 15分間加熱還流した。その後、 加熱を停止して室温で放冷し、核コロイドを形成させた。この核コロイド 26mlを、 500 mlの三口フラスコに入れ、液温が 30°Cになるまで恒温層内で撹拌した。液温が安定 したら、塩化金酸四水和物 0. 076gを溶解させた塩化金酸溶液 252mlと、 L ァスコ ルビン酸ナトリウム 0. 092gを溶解させた Lーァスコルビン酸溶液 256mlとを同時に 滴下して、 1時間撹拌しながら反応させて、平均粒径 40nmの金コロイドを形成させた Gold colloid with an average particle size of 40 nm was formed by the following method. Chloroauric acid tetrahydrate 0.17 g and trisodium citrate dihydrate 0 · 49 g were dissolved in 25 ml and 100 ml of ultrapure water, respectively, to prepare a chloroauric acid solution and a citrate solution. . Next, 6 ml of chloroauric acid solution and 200 ml of pure water were put into a 500 ml triloflasco and heated to reflux for 30 minutes. After the liquid temperature was stabilized, 50 ml of citrate solution was mixed and heated to reflux for 15 minutes. Thereafter, heating was stopped and the mixture was allowed to cool at room temperature to form a nuclear colloid. 26 ml of this nuclear colloid was placed in a 500 ml three-necked flask and stirred in the thermostatic layer until the liquid temperature reached 30 ° C. When the liquid temperature is stable, 252 ml of chloroauric acid solution in which 0.076 g of chloroauric acid tetrahydrate is dissolved and 256 ml of L-ascorbic acid solution in which 0.092 g of sodium L-ascorbate is dissolved are added dropwise at the same time. , Reacted for 1 hour with stirring to form colloidal gold particles with an average particle size of 40 nm
〇 Yes
[0024] また、平均粒径 80nmの金コロイドは、上記により得られた平均粒径 40nmの金コロ イドを用いて形成させた。平均粒径 40nmの金コロイド 33. 3mlを 500mlの三ロフラ スコに入れ、液温が 30°Cになるまで恒温層内で撹拌した。液温が安定したら、塩化 金酸四水和物 0. 039gを溶解させた塩化金酸溶液 118mlと、 L ァスコルビン酸ナ トリウム 0. 043gを溶解させた L ァスコルビン酸溶液 120mlとを同時に滴下して、 1 時間撹拌しながら反応させた。 [0024] A gold colloid having an average particle diameter of 80 nm was formed using the gold colloid having an average particle diameter of 40 nm obtained as described above. 33.3 ml of colloidal gold having an average particle size of 40 nm was placed in a 500 ml tri-lasco and stirred in the thermostatic layer until the liquid temperature reached 30 ° C. When the liquid temperature is stable, 118 ml of chloroauric acid solution in which 0.039 g of chloroauric acid tetrahydrate is dissolved and 120 ml of L ascorbic acid solution in which 0.043 g of sodium lascorbate is dissolved are added dropwise simultaneously. , 1 The reaction was allowed to stir for an hour.
[0025] lOOnmの金コロイドは、平均粒径 40nmの金コロイド 33. 4mlに対し、塩化金酸四 水和物 0. 079gを溶解させた塩化金酸溶液 247mlと、 L—ァスコルビン酸ナトリウム 0[0025] The colloidal gold of lOOnm is composed of 247ml of chloroauric acid solution in which 0.079g of chloroauric acid tetrahydrate is dissolved in 33.4ml of gold colloid with an average particle size of 40nm, and sodium L-ascorbate.
• 092gを溶解させた L—ァスコルビン酸溶液 251mlとを滴下したこと以外は、平均粒 径 80nmの金コロイドと同様の方法によって形成させた。 • It was formed by the same method as colloidal gold with an average particle size of 80 nm, except that 251 ml of L-ascorbic acid solution in which 092 g was dissolved was dropped.
[0026] [リンカ一の修飾] [0026] [Modification of linker]
上記方法により形成した粒径が 40、 80、 lOOnmの金コロイドに、炭素数 n nや、  The gold particle colloid formed by the above method with a particle size of 40, 80, lOOnm,
1、 2 置換基 R、Rの異なる第 1の化合物及び第 2の化合物を含むリンカ  1, 2 Substituents R, R A linker containing different first and second compounds
1 2 一を修飾させた 1 2 Modified one
。第 1実施形態〜第 4実施形態のそれぞれについて、金コロイドの粒径、リンカ一とし て用いた第 1の化合物及び第 2の化合物の種類、第 1の化合物と第 2の化合物とを含 む割合のモル比を、表 1に示す。 . Each of the first to fourth embodiments includes the particle size of the gold colloid, the types of the first compound and the second compound used as the linker, and the first compound and the second compound. The molar ratio is shown in Table 1.
[0027] 第 1実施形態は、粒径 80nmの金コロイドに、化 1の化合物（ジスルフイド基を有する )のみを含むリンカ一を修飾させたものと、化 2の化合物（チオール基を有する）のみ を含むリンカ一を修飾させたものとした。第 2実施形態は、炭素数 nが 3、 5、 7、 10で ある第 1の化合物を含むリンカ一を修飾させたものとした。第 3実施形態では、金コロ イドの粒径が 40nm、 80nm、 lOOnmである場合において、第 1の化合物と第 2の化 合物とのモル比を 10 : 0、 5 : 5、 1 : 9としたリンカ一を修飾させた。第 4実施形態は、第 1の化合物のみを含むリンカ一を金コロイドに修飾させたものであり、炭素数 n力 S 10 である第 1の化合物と、炭素数 nが 3、 5、 7、 10である第 1の化合物とを、モル比で 1 ： 9の割合で含むリンカ一を用いた。  [0027] In the first embodiment, a colloidal gold having a particle size of 80 nm is modified with a linker containing only a compound of formula 1 (having a disulfide group) and only a compound of formula 2 (having a thiol group). A linker containing 1 was modified. In the second embodiment, the linker including the first compound having a carbon number n of 3, 5, 7, 10 is modified. In the third embodiment, when the particle size of the gold colloid is 40 nm, 80 nm, or lOOnm, the molar ratio of the first compound to the second compound is 10: 0, 5: 5, 1: 9. The linker I was modified. In the fourth embodiment, a linker containing only the first compound is modified with a colloidal gold, and the first compound having a carbon number n force S 10 and the carbon number n is 3, 5, 7, A linker containing 10 first compound at a molar ratio of 1: 9 was used.
[0028] [表 1] [0028] [Table 1]
金コロイド 第 1の化合物 (A) 第 2の化合物 (B) モル比 粒柽 (A:B) 第 1実揄形據 HOOC(CH)10-S OH(CH)2-S Gold colloid 1st compound (A) 2nd compound (B) Molar ratio Granules (A: B) 1st real bowl HOOC (CH) 10 -S OH (CH) 2 -S
1-1 I I  1-1 I I
80nm HOOC(CH)10-S OH(CH)2-S 1:980nm HOOC (CH) 10 -S OH (CH) 2 -S 1: 9
1-2 HOOC(CH)10-SH OH(CH)2-SH 1-2 HOOC (CH) 10 -SH OH (CH) 2 -SH
第 2実施形態 2-1  Second Embodiment 2-1
2-2 HOOC(CH)n-S n= 7 OH(CH)2-S 2-2 HOOC (CH) n -S n = 7 OH (CH) 2 -S
80 I I 1:9 80 I I 1: 9
2-3 HOOC(CH)„-S n= 5 OH(CH)2-S 2-3 HOOC (CH) „-S n = 5 OH (CH) 2 -S
2-4 n= 3  2-4 n = 3
第 3実施形態 3-1 10:0  Third embodiment 3-1 10: 0
HOOC(CH)10-S OH(CH)2-S HOOC (CH) 10 -S OH (CH) 2 -S
3-2 40nm I I 5:5  3-2 40nm I I 5: 5
HOOC(CH)10-S OH(CH)2-S HOOC (CH) 10 -S OH (CH) 2 -S
3-3 1 :9 3-3 1: 9
3-4 10:0 3-4 10: 0
HOOC(CH)10-S OH(CH)2-S HOOC (CH) 10 -S OH (CH) 2 -S
3-5 80nm I I 5:5  3-5 80nm I I 5: 5
HOOC(CH)t0-S II OH(CH)2-S HOOC (CH) t0 -S II OH (CH) 2 -S
3-6 1 :9  3-6 1: 9
O  O
3-7 10:0  3-7 10: 0
HOOC(CH)10-S OH(CH)2-S HOOC (CH) 10 -S OH (CH) 2 -S
3-8 lOOnm I I 5:5  3-8 lOOnm I I 5: 5
HOOC(GH)10-S OH(CH)2-S HOOC (GH) 10 -S OH (CH) 2 -S
3-9 1 :9  3-9 1: 9
Figure imgf000010_0001
Figure imgf000010_0001
[0029] 具体的なリンカ一の修飾方法について、第 3実施形態におけるモル比 5 :5の金コロ イドを例として説明する。上記方法により得られた金コロイド 100mlに、リンカ一である 第 1の化合物のメタノール溶液 5ml (濃度 10 M)と、第 2の化合物のメタノール溶液 5ml (濃度 10 M)とを添加し、室温で 24時間反応させた。その後、 10mM ホウ酸 緩衝液 (pH 8. 1)1Lを用いて、 5時間の透析を 3回行って、未反応の溶液を取り除 き、リンカ一を修飾した金コロイドを得た。その他の実施形態についても、それぞれ上 記と同様の方法によりリンカ一の修飾を行った。 [0029] A specific method for modifying the linker will be described with reference to a gold colloid having a molar ratio of 5: 5 in the third embodiment as an example. To 100 ml of gold colloid obtained by the above method, add 5 ml of methanol solution (concentration 10 M) of the first compound, which is the linker, and 5 ml of methanol solution (concentration 10 M) of the second compound, at room temperature. The reaction was performed for 24 hours. Thereafter, dialysis was performed 3 times for 5 hours using 1 L of 10 mM borate buffer (pH 8.1) to remove the unreacted solution to obtain a colloidal gold modified with a linker. In the other embodiments, the linker was modified by the same method as above.
[0030] [抗体の固定]  [0030] [Immobilization of antibody]
上記方法により、粒径及びリンカ一が異なるそれぞれの金コロイドについて、抗体を 固定させた。抗体には、妊娠の成立を診断するために用いる絨毛性性腺刺激ホルモ ン抗体を用いた。リンカ一を修飾した金コロイド 900 ^ 1に、 0. 04mM 1ーェチルー 3— (3—ジメチルァミノプロピル）カルボキシルイミド塩酸塩 100mlを加えて 10分間 反応させて、 100 g/ml絨毛性性腺刺激ホルモン抗体（抗ヒトモノクローナル抗体 clone5008、 Biochemica社製） 100 1をカロえ、 3時間室温で反応させた。尚、力 ルポキシルイミド塩酸塩と、絨毛性性腺刺激ホルモン抗体には、緩衝液として 10mM ホウ酸緩衝液 (ρΗ8 · 1 )を使用した。 By the above method, the antibody was immobilized on each gold colloid having a different particle diameter and linker. Antibodies include chorionic gonad-stimulating hormone used to diagnose the establishment of pregnancy Antibody was used. Linker-modified gold colloid 900 ^ 1 with 0.04 mM 1-ethyl-3- (3-dimethylaminopropyl) carboximide hydrochloride 100 ml and allowed to react for 10 minutes, 100 g / ml chorionic gonadotropin The antibody (anti-human monoclonal antibody clone5008, manufactured by Biochemica) 100 1 was prepared and reacted at room temperature for 3 hours. In addition, 10 mM borate buffer solution (ρΗ8 · 1) was used as a buffer solution for force lupoxylimide hydrochloride and chorionic gonadotropin antibody.
[0031] その後、 1 % ポリエチレングリコーノレ 20000 (50mM リン酸カリウム緩衝液（pH7 . 5)を使用した。）を 50〃1と、 10% ゥシ血清アルブミン（以下、 BSAという。 : 50m M リン酸カリウム緩衝液（pH9. 0)を使用した。） 100 1を加え、さらに 10分間反応 させた。その後、 4°Cにおいて、 15分間遠心分離を行い、上澄みの 9割程度を除去し て、沈降した抗体固定金コロイドを採取した。採取した沈殿は、超音波分散させた後 、保存液（1. 0% BSA、 0. 05% ポリエチレングリコール 20000、 0. 1 % NaN 、 [0031] After that, 1% polyethylene glycolol 20000 (50 mM potassium phosphate buffer (pH 7.5) was used) was 50-1 and 10% urine serum albumin (hereinafter referred to as BSA): 50 mM phosphorus Potassium acid buffer solution (pH 9.0) was used.) 100 1 was added, and the mixture was further reacted for 10 minutes. Thereafter, the mixture was centrifuged at 4 ° C for 15 minutes, and about 90% of the supernatant was removed, and the precipitated antibody-fixed gold colloid was collected. The collected precipitate was ultrasonically dispersed and then stored in a storage solution (1.0% BSA, 0.05% polyethylene glycol 20000, 0.1% NaN,
3 Three
150mM NaCl、 20mM Tris-HCl (pH8. 2) ) 900〃 1を加えた。この遠心分離 による洗浄を、 3回繰り返し行った。尚、遠心分離は、金コロイドの粒径が 40nmの場 合 3000 X g、 80腹及び 100應の場合 1500 X gの条件で行った。洗浄後の抗体 固定金コロイドは、分光光度計で吸光度を測定し、波長 520〜600nmの間における 吸光度の最大値が 6となるよう、保存液を用いて調整を行った。 150 mM NaCl, 20 mM Tris-HCl (pH 8.2)) 900 1 was added. This washing by centrifugation was repeated three times. Centrifugation was performed under the conditions of 3000 X g when the colloidal gold particle size was 40 nm, and 1500 X g when the particle size was 80 and 100. The antibody-immobilized gold colloid after washing was measured for absorbance with a spectrophotometer, and was adjusted using a stock solution so that the maximum absorbance at a wavelength of 520 to 600 nm was 6.
[0032] 比較として、リンカ一を修飾していない金コロイドについて、抗体固定金コロイドを作 成した。平均粒径 40nm、 80nm、 lOOnmの金コロイドを用いて、 50mM KH PO [0032] For comparison, an antibody-immobilized gold colloid was prepared for a gold colloid in which the linker was not modified. 50mM KH PO using gold colloid with average particle size of 40nm, 80nm, lOOnm
2 4 twenty four
(pH7. 5) 100 1と、 100 8/1111絨毛性性腺刺激ホルモン抗体（5111^ [ KH PO (pH7.5) 100 1 and 100 8/1111 chorionic gonadotropin antibody (5111 ^ [KH PO
2 4 twenty four
(pH7. 5) ) 100 1とを添加して 10分間反応させた以外は、上記と同様の方法により 、抗体固定金コロイドを作成した。 (pH 7.5)) An antibody-immobilized gold colloid was prepared in the same manner as described above except that 100 1 was added and allowed to react for 10 minutes.
[0033] [テストストリップの作成] [0033] [Create test strip]
ィムノクロマトグラフィーによる診断のための、テストストリップを作成した。移動層で あるメンブレン上に、抗原の有無を判定するためのテストラインと、金コロイド力 メン ブレン上で正常に発色しているかどうかの目安となるコントロールラインとを作成した 。テストラインには、抗原と複合体を形成した抗体固定金コロイドのみを捕捉する抗体 を塗布し、コントロールラインには、抗原を含まない抗体固定金コロイドを捕捉する抗 体を塗布した。 Test strips were made for immunochromatographic diagnosis. A test line for determining the presence / absence of an antigen on a membrane serving as a moving layer and a control line serving as a guideline for whether or not normal color was developed on a colloidal gold membrane were prepared. The test line is coated with an antibody that captures only the antibody-immobilized gold colloid that forms a complex with the antigen, and the control line is an antibody that captures the antibody-immobilized gold colloid that does not contain the antigen. The body was applied.
[0034] テストラインは、抗体として抗 h a S抗体を用いた溶液 (表 2)を調整し、この溶液 0. 7 5〃1を、定量分注装置（Biojet Quanti 3000、 BioDot社製）により、 50mm/秒 、 20. 83nL/dot、 0. 28mmピッチの条件で、メンブレンに塗布して作成した。尚、 メンブレンとしては、ニトロセルロース製で、幅 25mm、長さ 30cmのものを使用した。  [0034] The test line was prepared by preparing a solution (Table 2) using an anti-ha S antibody as an antibody, and using this solution 0.7 5 1 by a quantitative dispensing device (Biojet Quanti 3000, manufactured by BioDot). It was prepared by applying to a membrane under the conditions of 50 mm / second, 20.83 nL / dot, and 0.28 mm pitch. The membrane was made of nitrocellulose, 25 mm wide and 30 cm long.
[0035] [表 2]  [0035] [Table 2]
Figure imgf000012_0001
Figure imgf000012_0001
※抗ヒト卵胞刺激ホルモン (LH)モノクローナル抗体（aサブユニット、クローン 6601 )  * Anti-human follicle-stimulating hormone (LH) monoclonal antibody (a subunit, clone 6601)
[0036] コントロールラインは、抗体として抗マウス抗体を用いた溶液 (表 3)を調整し、テスト ラインと同様の塗布条件で、メンブレン上に作成した。 [0036] A control line was prepared on a membrane by preparing a solution (Table 3) using an anti-mouse antibody as an antibody, under the same coating conditions as the test line.
[0037] [表 3]
Figure imgf000012_0002
[0037] [Table 3]
Figure imgf000012_0002
[0038] テストライン及びコントロールラインを形成したメンブレンは、インキュベータで、 42 °Cにおいて 1時間乾燥させた後、室温でさらに 2時間乾燥させた。その後、メンブレン を、抗体固定金コロイドがニトロセルロース膜へ吸着することを防ぐためのブロッキン グ溶液に浸漬した。ブロッキング溶液は、 0. 5%カセイン（50mM ホウ酸緩衝液 (p H8. 5) )とし、メンブレンのテストライン側を先に浸漬し、その後に全体を浸漬して、 室温で 30分静置した。その後、ブロッキング溶液からメンブレンを取り出し、余分な試 薬を除いた後、洗浄液である 0. 01 % ドデシル硫酸ナトリウム（5mM リン酸緩衝液 (pH7. 5) )に、前記と同様に浸漬し、 30分静置した。洗浄液からメンブレンを取り出 し、 24時間静置した後、吸収パッドを貼り付けて、 4mm幅に切断した。  [0038] The membrane on which the test line and the control line were formed was dried in an incubator at 42 ° C for 1 hour, and further dried at room temperature for 2 hours. The membrane was then immersed in a blocking solution to prevent the antibody-immobilized gold colloid from adsorbing to the nitrocellulose membrane. The blocking solution was 0.5% casein (50 mM borate buffer (pH 8.5)), the membrane was soaked first, and then the whole was immersed and allowed to stand at room temperature for 30 minutes. . After removing the membrane from the blocking solution and removing the excess reagent, the membrane was immersed in 0.01% sodium dodecyl sulfate (5 mM phosphate buffer (pH 7.5)) as a washing solution as described above. Left to stand. The membrane was taken out from the cleaning solution and allowed to stand for 24 hours, and then an absorbent pad was attached and cut to a width of 4 mm.
[0039] [ィムノクロマトグラフィー]  [0039] [Immuno Chromatography]
マイクロプレートに、抗原であるリコンピナント hCG (l %BSA、 50mM リン酸緩衝 液（ρΗ7· 4) )を 40 1と、抗体固定金コロイド 4 1とを投入して混合した後、テストスト リップを差し込み展開させた。 Into the microplate, add the antigen recombinant hCG (l% BSA, 50 mM phosphate buffer (ρΗ7.4)) 40 1 and antibody-immobilized gold colloid 4 1 and mix them. A lip was inserted and developed.
[0040] [発色強度の測定]  [0040] [Measurement of color intensity]
ィムノクロマトグラフィーを行った後、メンブレン上のテストラインにおいて、金コロイド の発色強度を測定した。測定には、反応ライン自動読み取り機器 (Quad Scan : Bio Dot社製)を用いた。第 1実施形態〜第 4実施形態のリンカ一を修飾した金コロイド の発色強度について、リンカ一を修飾しなかった金コロイドの発色強度に対する比率 を算出した結果を図;!〜 4のグラフに示す。  After immunochromatography, the color intensity of colloidal gold was measured on a test line on the membrane. For the measurement, a reaction line automatic reading device (Quad Scan: manufactured by Bio Dot) was used. Figures 1 to 4 show the results of calculating the ratio of the colloidal intensity of gold colloid modified with the linker of the first to fourth embodiments to the intensity of colloidal gold without modification of the linker. .
[0041] 図 1より、第 1の化合物と第 2の化合物とを、モル比で 1 : 9の割合で含むリンカ一を 用いた金コロイドによれば、化 1のみを用いた場合であっても、化 2のみを用いた場合 であっても、リンカ一を修飾しなかった金コロイドに対する発色強度の比率が 100% 以上となり、充分な発色を示す金コロイドとなることが分かった。また、図 2より、第 1の 化合物の炭素数 nが 5、 7、 10である場合には、 n力 ¾の場合に比べて発色強度の 高レ、ものとなることが分かった。  [0041] From FIG. 1, according to the gold colloid using the linker containing the first compound and the second compound in a molar ratio of 1: 9, it is a case where only chemical 1 is used. However, even when only chemical formula 2 was used, it was found that the ratio of the color development intensity to the gold colloid that was not modified with the linker was 100% or more, and the gold colloid exhibiting sufficient color development. Further, from FIG. 2, it was found that when the carbon number n of the first compound is 5, 7, and 10, the coloring intensity is higher than that in the case of n force.
[0042] 図 3より、粒径 40、 80、 lOOnmのいずれの金コロイドを用いたにも、リンカ一を修飾 しなかった金コロイドに対する発色強度の比率が 100%以上となり、良好な発色が示 される金コロイドとなることが分かった。また、第 1の化合物と第 2の化合物との割合の モル比は、粒径 40nmの場合は 5： 5 (すなわち 1： 1)、粒径 80nm及び lOOnmの場 合には 1： 9であると、金コロイドの発色強度がより高いものとなることが分かった。  [0042] From Fig. 3, the ratio of the color intensity to the gold colloid without modification of the linker is 100% or more regardless of which gold colloid having a particle size of 40, 80, or lOOnm is used, indicating good color development. Turned out to be a colloidal gold. The molar ratio of the ratio of the first compound to the second compound is 5: 5 (ie 1: 1) for a particle size of 40 nm, and 1: 9 for a particle size of 80 nm and lOOnm. It was found that the color intensity of the colloidal gold was higher.
[0043] そして、図 4より、第 1の化合物のみを用いた場合の金コロイドは、リンカ一を修飾し なかった金コロイドに対する発色強度の比率が 100%程度であり、発色強度は高くな りにくい傾向であった力 リンカ一の炭素数 nが大きくなるほど発色強度が大きくなる 関係となり、発色強度を調整しやすいことが分かった。  [0043] From FIG. 4, the gold colloid when only the first compound is used has a color development intensity ratio of about 100% with respect to the gold colloid not modified with the linker, and the color development intensity is high. The force that was difficult to develop The relationship was that the color development intensity increased as the number of carbon atoms n of the linker increased, and it was found that the color development intensity was easy to adjust.
産業上の利用可能性  Industrial applicability
[0044] 以上で説明したように、本発明の金コロイドは、体外診断薬として好適な金コロイド となる。また、発色の強度が大きぐ微量の抗原であっても検出可能であり、広範な粒 径において発色性が良好なものとなる。 [0044] As described above, the gold colloid of the present invention is a gold colloid suitable as an in vitro diagnostic agent. Further, even a trace amount of antigen with high color development intensity can be detected, and the color developability is good over a wide range of particle sizes.

Claims

請求の範囲 The scope of the claims
[1] 金コロイドと、前記金コロイドを修飾し抗体を固定するためのリンカ一と、を含む体外 診断薬用金コロイドであって、  [1] An in vitro diagnostic gold colloid comprising gold colloid and a linker for immobilizing the gold colloid and fixing the antibody,
前記リンカ一は、化 1及び/又は化 2で示される化合物からなり、置換基 R、 R及  The linker is composed of a compound represented by Chemical Formula 1 and / or Chemical Formula 2, and has substituents R, R and
1 2 び/又は炭素数 n、 nが異なる 2種以上の化合物からなる体外診断薬用金コロイド。  1 2 and / or gold colloid for in-vitro diagnostics consisting of two or more compounds with different n and n carbon numbers.
1 2  1 2
[化 1コ
Figure imgf000014_0001
Figure imgf000014_0002
[Chemical 1
Figure imgf000014_0001
Figure imgf000014_0002
(ni=2〜10，
Figure imgf000014_0003
OH, CHO, NH2) (ni = 2-10,
Figure imgf000014_0003
(OH, CHO, NH2)
[化 2] [Chemical 2]
I^CHwrk― SH I ^ CHwrk― SH
(112=2〜； 10、 R2=COOH， OH, CHO, NH2) (112 = 2 ~; 10, R 2 = COOH, OH, CHO, NH2)
[2] リンカ一は、化 1又は化 2で示される化合物のいずれかであって、置換基 R、 R及 [2] The linker is one of the compounds represented by Chemical Formula 1 or Chemical Formula 2 and has substituents R, R and
1 2 び/又は炭素数 n、nが異なる 2種以上の化合物からなる請求項 1に記載の体外診  The in vitro examination according to claim 1, comprising 1 2 and / or two or more compounds having different n and n carbon numbers.
1 2  1 2
断薬用金コロイド。  Colloidal gold colloid.
[3] リンカ一は、置換基 R又は Rがカルボキシル基である第 1の化合物と、置換基 R又  [3] The linker includes the first compound in which the substituent R or R is a carboxyl group, the substituent R or R
1 2 1 は Rがカルボキシル基以外の置換基である第 2の化合物とを含む請求項 1又は請求 1 2 1 includes a second compound in which R is a substituent other than a carboxyl group.
2 2
項 2に記載の体外診断薬用金コロイド。  Item 3. An in vitro diagnostic gold colloid according to Item 2.
[4] 第 2の化合物の置換基 R又は Rは、ヒドロキシル基である請求項 3に記載の体外 [4] The extracorporeal body according to claim 3, wherein the substituent R or R of the second compound is a hydroxyl group.
1 2  1 2
診断薬用金コロイド。  Gold colloid for diagnostics.
[5] 第 1の化合物の炭素数 n又は nと、第 2の化合物の炭素数 n又は nとの差が 2以  [5] The difference between the carbon number n or n of the first compound and the carbon number n or n of the second compound is 2 or more.
1 2 1 2  1 2 1 2
上である請求項 3又は 4に記載の体外診断薬用金コロイド。  The colloidal gold colloid for in vitro diagnostics according to claim 3 or 4, which is above.
[6] 第 1の化合物は、炭素数 n又は n力 〜； 10の化合物であり、第 2の化合物は、炭素 数 n又は n力 〜8の化合物である請求項 3〜請求項 5のいずれかに記載の体外診[6] The first compound is a compound of carbon number n or n force ˜; 10 and the second compound is carbon The in vitro examination according to any one of claims 3 to 5, wherein the compound is a compound having a number n or n force -8.
1 2 1 2
断薬用金コロイド。  Colloidal gold colloid.
[7] リンカ一は、第 1の化合物と第 2の化合物とを、モル比で 1： 1〜0. 1： 9. 9の割合で 含むものである請求項 3〜請求項 6のいずれかに記載の体外診断薬用金コロイド。  [7] The linker according to any one of claims 3 to 6, wherein the linker contains the first compound and the second compound in a molar ratio of 1: 1 to 0.1: 9.9. Gold colloid for in vitro diagnostics.
[8] 金コロイドの粒径は、 10nm〜150nmである請求項 1〜7のいずれかに記載の体 外診断薬用金コロイド。 [8] The gold colloid for in vitro diagnostics according to any one of claims 1 to 7, wherein the gold colloid has a particle size of 10 nm to 150 nm.
[9] 試料を展開するための移動層と、 [9] A moving bed for spreading the sample,
前記移動層の一端に設けられ、抗原に対応する抗体が固定された金コロイドと、 前記移動層の他方の端部に含まれる抗原の有無を判定するための部分と、を備え るィムノクロマトグラフィー用診断キットであって、  An immunochromatography comprising: a gold colloid provided at one end of the moving layer, to which an antibody corresponding to an antigen is fixed; and a portion for determining the presence or absence of an antigen contained at the other end of the moving layer. A diagnostic kit for lithography,
前記金コロイドは、請求項 1〜8のいずれかに記載の金コロイドであるィムノクロマト グラフィー用診断キット。  The immunochromatography diagnostic kit according to claim 1, wherein the colloidal gold is the colloidal gold according to claim 1.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06116602A (en) * 1990-12-05 1994-04-26 Akzo Nv Ligand-gold bond
JP2003248001A (en) * 2002-02-22 2003-09-05 Inst Of Physical & Chemical Res Method for screening protein having intermolecular interaction with ligand
JP2003536074A (en) * 2000-06-07 2003-12-02 エフ．ホフマン−ラ ロシュ アーゲー Core-shell metal particles coated with dextran or aminodextran bearing SH groups, and uses thereof
JP2004170367A (en) * 2002-11-22 2004-06-17 Inst Of Physical & Chemical Res Manufacturing method for nucleic acid microarray and nucleic acid spotting solution
JP2006266832A (en) * 2005-03-23 2006-10-05 Fuji Photo Film Co Ltd Fixation method, biosensor, and test method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06116602A (en) * 1990-12-05 1994-04-26 Akzo Nv Ligand-gold bond
JP2003536074A (en) * 2000-06-07 2003-12-02 エフ．ホフマン−ラ ロシュ アーゲー Core-shell metal particles coated with dextran or aminodextran bearing SH groups, and uses thereof
JP2003248001A (en) * 2002-02-22 2003-09-05 Inst Of Physical & Chemical Res Method for screening protein having intermolecular interaction with ligand
JP2004170367A (en) * 2002-11-22 2004-06-17 Inst Of Physical & Chemical Res Manufacturing method for nucleic acid microarray and nucleic acid spotting solution
JP2006266832A (en) * 2005-03-23 2006-10-05 Fuji Photo Film Co Ltd Fixation method, biosensor, and test method

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