WO2011034115A1 - アビジンとビオチン誘導体の解離方法及び解離剤 - Google Patents
アビジンとビオチン誘導体の解離方法及び解離剤 Download PDFInfo
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- WO2011034115A1 WO2011034115A1 PCT/JP2010/066005 JP2010066005W WO2011034115A1 WO 2011034115 A1 WO2011034115 A1 WO 2011034115A1 JP 2010066005 W JP2010066005 W JP 2010066005W WO 2011034115 A1 WO2011034115 A1 WO 2011034115A1
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- derivative
- avidin
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
Definitions
- the present invention relates to a dissociation method and dissociator for avidin or streptavidin and a biotin derivative.
- Avidin or streptavidin has a strong specific affinity with biotin or a biotin derivative, and can easily be bound (mixed between avidin and biotin) when mixed under physiological conditions.
- a binding substance or a binding between avidin or the like and a biotin derivative is collectively referred to as “ABC binding”) to form a conjugate of avidin or the like and biotin to form a target substance such as a cell (“target A technique for separating the material is also used (Patent Document 1).
- an ABC bond is formed by mixing a biotin-labeled probe molecule in which biotin is previously bound to a probe molecule such as an antibody having binding properties with a target substance and an insoluble carrier on which avidin is immobilized under physiological conditions. If the target substance can be captured using this capture carrier, and then the ABC binding or the binding between the probe molecule and the target substance can be cleaved, the target can be captured. It is possible to separate substances (Patent Document 1).
- Patent Document 2 a method using a biotin derivative having a lower affinity for avidin or the like than biotin
- a method using an avidin variant having a lower affinity for biotin than avidin is known.
- the above-mentioned capture carrier is prepared using a biotin derivative such as desthiobiotin (Patent Document 2) having a lower affinity for avidin or the like than biotin, and after capturing the target substance, a large amount of biotin
- Patent Document 2 a method of cleaving ABC binding and separating a target substance by adding.
- biotin is considered to function as a competitive inhibitor for the binding of desthiobiotin and avidin.
- biotin is considered to function as a competitive inhibitor for the binding of desthiobiotin and avidin.
- it is difficult to efficiently cleave ABC bonds, and it is difficult to efficiently separate target substances.
- the present invention provides a method for efficiently dissociating avidin or the like and a biotin derivative having an affinity lower than that of biotin in a short time under mild conditions and a dissociator thereof.
- the present inventors surprisingly found a water-soluble polymer to which biotin and the like are bound.
- a dissociating agent it was found that avidin and the like and a biotin derivative having a lower affinity than biotin were dissociated extremely efficiently under mild conditions, and the present invention was completed.
- the present invention relates to a conjugate of biotin derivative (1) having a lower affinity for avidin and avidin or a conjugate of streptavidin and biotin derivative (1) (hereinafter collectively referred to as “avidin-biotin complex”). And a biosoluble derivative (1) are dissociated with a biotin or a derivative thereof (2) and a water-soluble polymer (hereinafter referred to as “dissociator”). A method is provided.
- the present invention also provides a method for separating a target substance, (A) a step of mixing a capture carrier and a target substance in an aqueous solvent to obtain a mixed solution 1; (B) a step of mixing the liquid mixture 1 with a water-soluble polymer to which biotin or a derivative thereof (2) is bound to obtain a liquid mixture 2; (C) A method including the step of separating the target substance from the mixed solution 2 is provided.
- the capture carrier is a combination of the following (A) and (B).
- Affinity immobilized on a substance having specific affinity for a target substance hereinafter referred to as “probe molecule” is higher than that of biotin.
- the present invention provides a dissociator used for dissociating avidin or streptavidin and biotin derivative (1), which contains a water-soluble polymer to which biotin or a derivative thereof (2) is bound.
- the bond between avidin and the biotin derivative (1) can be efficiently cleaved in a short time under mild conditions to dissociate the avidin and the biotin derivative (1). Further, by applying the method to the separation of the target substance, the target substance obtained by binding to the capture carrier can be easily separated. Even when the target substance is unstable with respect to temperature conditions and pH conditions such as proteins and cells, the target substance can be separated while maintaining its activity by using the method of the present invention.
- the method for dissociating avidin and the like and the biotin derivative (1) according to an embodiment of the present invention includes a step of mixing an avidin-biotin complex and a water-soluble polymer to which biotin or a derivative thereof (2) is bound.
- the dissociation method of the present invention is usually performed in a solvent containing water as a main component.
- the avidin-biotin complex is preferably dissolved in the solvent.
- the avidin-biotin complex is contained in the solvent together with the carrier. May be dispersed.
- the avidin-biotin complex used in the present invention is a conjugate of avidin or streptavidin and a biotin derivative (3) having a lower affinity for avidin than biotin (a compound represented by the following formula (3)).
- avidin or streptavidin commercially available products can be used, and these may be those obtained by separating and purifying those existing in nature, or artificially produced by genetic engineering techniques. It may be an avidin or a streptavidin derivative modified within a range not losing its ability to bind to biotin. Examples of the derivatives of avidin or streptavidin include chemically modified avidin or streptavidin such as succinylation, and a monomer of avidin or streptavidin which is usually a tetramer.
- the biotin derivative (1) is not particularly limited as long as it is a biotin derivative having a lower affinity for avidin than biotin.
- Avidin and biotin derivative (1) are effectively dissociated by mixing the avidin-biotin complex with a water-soluble polymer to which biotin or its derivative (2) is bound due to its lower affinity for avidin. can do.
- Examples of such a biotin derivative (1) include desthiobiotin (a compound represented by the following formula (4)), 2-iminobiotin (a compound represented by the following formula (5)), 3,4- Derivatives that differ from biotin in the cyclic structure portion of biotin such as diaminobiotin (compound represented by the following formula (6)), and chemically modified products thereof.
- a lower affinity for avidin than biotin means that the binding constant is one or more orders of magnitude smaller than that of avidin and biotin (10 15 M).
- the binding constant between the compounds represented by the above formulas (4) to (6) and avidin is 10 6 M to 10 13 M.
- the avidin-biotin complex used in the present invention may be immobilized on an insoluble carrier via avidin or the like. Since the avidin-biotin complex is immobilized on an insoluble carrier, the avidin-biotin complex can be easily separated from the dispersion containing the avidin-biotin complex by a method such as centrifugation or filtration.
- the insoluble carrier used for immobilizing avidin and the like is not particularly limited in shape, and examples thereof include organic or inorganic particles, a flat substrate, a substrate having a fine channel, a microwell plate, and the like.
- the organic particles are preferably particles made of an organic synthetic polymer mainly composed of polystyrene having an average particle size of 0.5 to 10 ⁇ m.
- the particles are preferably magnetic particles in that they can be separated by magnetism. Examples of commercially available magnetic particles include Dynabeads M-450 Tosylactivated (manufactured by Invitrogen) and Magnosphere MS300 / Carboxyl (manufactured by JSR Corporation).
- the magnetic particles are preferably produced according to a known method (for example, see Japanese Patent Publication No. 05-010808 or Japanese Patent Application Laid-Open No. 2007-288133), and can reduce nonspecific adsorption of proteins and cells. .
- the method for immobilizing an insoluble carrier such as avidin is not particularly limited, and a known method (for example, see JP-A-2001-158800) can be used.
- a known method for example, see JP-A-2001-158800
- an amide bond is formed by reacting an amino group in a molecule such as avidin with a carboxyl group on the carrier surface in the presence of a dehydrating condensation agent such as water-soluble carbodiimide.
- a dehydration condensing agent may be reacted in advance with the carboxyl group of the carrier, and then avidin or streptavidin may be added and reacted.
- the avidin-biotin complex used in the present invention may be a conjugate of the following (A) and (B) (hereinafter referred to as “capture carrier”).
- the probe molecule has a property capable of specifically binding to the target substance and is bound to the biotin derivative (1).
- the probe molecule and the biotin derivative (1) may be directly bonded or may be bonded via a spacer.
- the biotin derivative (1) immobilized on the probe molecule is a biotin derivative (1) having a lower affinity for avidin than the biotin described in the section on avidin-biotin complex.
- the method for binding the biotin derivative (1) to the probe molecule is not particularly limited.
- a method similar to the method for immobilizing avidin or the like on the surface of the carrier can be used.
- the amide bond is formed by reacting the carboxyl group in the biotin molecule with the amino group in the probe molecule in the presence of a dehydrating condensation agent such as water-soluble carbodiimide.
- a dehydrating condensation agent such as water-soluble carbodiimide.
- FIG. 1 shows a conceptual diagram of a capture carrier bound to a target substance.
- the target substance is not particularly limited.
- cells for example, various cells of bacteria, fungi, animals or plants
- viruses proteins (for example, various antigens or antibodies, enzymes), single-stranded or double-stranded
- nucleic acids such as DNA or RNA
- steroid lipids such as estrogen, glycolipids, polysaccharides and the like.
- target cells Specific examples of cells that are target substances (hereinafter referred to as “target cells”) are not particularly limited.
- normal cells for example, stem cells such as hematopoietic stem cells, blood cells such as leukocytes, etc.
- cancer cells For example, circulating cancer cells in peripheral blood derived from breast cancer and lung cancer, exfoliated cancer cells present in stool derived from colon cancer, cancer cells in menstrual blood derived from uterine cancer, Cancer cells derived from bladder cancer or the like present in urine.
- an avidin-biotin complex is mixed with a water-soluble polymer (hereinafter also referred to as “dissociator”) to which biotin or a derivative thereof (2) is bound.
- dissociator a water-soluble polymer
- the dissociator is most preferably a water-soluble polymer to which biotin is bound.
- biotin derivatives (2) that can be used in the dissociating agent include biocytin (biocytin: biotin- ⁇ -N-lysine; a compound represented by the following formula (7)), biotin-N-hydroxysuccinimide ester (see below).
- examples include derivatives different from biotin in the cyclic structure portion of biotin described in the section on avidin-biotin derivatives. Among these, for the cyclic structure portion of biotin, a derivative having the same structure as biotin is preferable because of its strong affinity with avidin.
- a dissociating agent may be added to and mixed with a liquid sample containing an avidin-biotin complex under physiological conditions.
- physiological conditions for example, conditions of 20 to 40 ° C., about 1 atm, pH 5 to 9 and the like are preferable.
- the dissociating agent of the present invention can be dissociated avidin or the like and the biotin derivative (1) efficiently.
- a dissociation agent is added to an aqueous sample containing an avidin-biotin complex
- avidin contained in the dissociation agent or a derivative thereof (2) competes with the biotin derivative (1) bound to avidin, etc.
- the biotin derivative (1) can be dissociated.
- the dissociation agent in which biotin or its derivative (2) is bound to the water-soluble polymer is more efficient than biotin or its derivative (2) that is not bound to the water-soluble polymer, although the reason is not necessarily clear.
- avidin and the biotin derivative (1) can be dissociated.
- the water-soluble polymer is not particularly limited as long as it is a water-soluble polymer and can bind to biotin or a derivative (2) thereof, but it binds to the water-soluble polymer using a carboxyl group possessed by biotin.
- Water-soluble polymers having functional groups such as amino groups, sulfhydryl groups, and carboxyl groups are preferred.
- Examples include organic synthetic polymers having reactive functional groups such as amino groups in addition to water-soluble proteins and polysaccharides.
- the water-soluble proteins for example, BSA (bovine serum albumin), etc. HSA (human serum albumin) are preferred.
- the polysaccharide include CMC (carboxymethyl cellulose) and chitosan.
- synthetic polymers such as PAA (polyacrylic acid) and polyallylamine, and polyamino acids such as polylysine and polyaspartic acid are preferable.
- water soluble when dissolved in a concentration of 1 mg / ml, refers to the property of dissolving without clouding.
- the biotin-binding water-soluble polymer is preferably 1,000 to 1,000,000.
- the avidin-biotin complex can be dissociated with high efficiency.
- the molecular weight of the biotin-conjugated water-soluble polymer can be measured, for example, by gel permeation chromatography.
- the average number of molecules of biotin or its derivative (2) bound per molecule of the water-soluble polymer is preferably as much as possible within the range in which the water-solubility is not lost. More preferably, it is more preferably 5 to 1,000, and particularly preferably 10 to 100.
- the more biotin or its derivative (2) bound per molecule of water-soluble polymer the higher the dissociation efficiency between avidin and the biotin derivative (1).
- the average value of the number of molecules of biotin or its derivative (2) bound per molecule of the water-soluble polymer can be measured by the method (HABA method) described in Examples described later.
- biotin or a derivative thereof (2) is reacted with a water-soluble polymer having at least one of a carboxyl group and an amino group.
- the method of obtaining is mentioned.
- the water-soluble polymer contains an amino group
- a method of dehydrating and condensing the amino group in the water-soluble polymer and the carboxyl group of biotin can be mentioned.
- the water-soluble polymer contains a carboxyl group
- a method of dehydrating and condensing the carboxyl group in the water-soluble polymer and the amino group of a biotin derivative having an amino group can be mentioned.
- the method for separating a target substance of the present invention comprises (a) a step of mixing a capture carrier and a target substance in an aqueous solvent to obtain a mixed liquid 1, and (b) a mixed liquid 1 and biotin or a derivative thereof (2). A step of mixing the water-soluble polymer to which is bound to obtain the mixed solution 2, and (c) a step of separating the target substance from the mixed solution 2.
- the capture carrier is (A) Avidin or streptavidin immobilized on an insoluble carrier and (B) a biotin derivative (2) immobilized on a substance having specific affinity for a target substance (hereinafter referred to as “probe molecule”) It is a combination.
- Step of obtaining the liquid mixture 1 by mixing the capture carrier and the target substance in an aqueous solvent This step is a step of binding the capture carrier and the target substance. Specifically, by mixing the capture carrier and the target substance, the probe molecule contained in the capture carrier and the target substance or a molecule having affinity with the probe molecule contained in the target substance are specifically bound. .
- the capturing carrier and the target substance are as described above.
- the aqueous solvent a solvent containing water having a pH of 5 to 9 as a main solvent is preferable, and a buffer solution such as a phosphate buffer or a Tris buffer is more preferable.
- the capturing carrier and the target substance In order to mix the capturing carrier and the target substance, it is preferable to mix the capturing carrier dispersed in the aqueous solvent and the target substance dissolved or dispersed in the same or different aqueous solvent. After mixing, it is preferable to mix at 10 to 40 ° C. for 1 to 60 minutes. By mixing under such conditions, the capture carrier and the target substance are efficiently bound.
- the amount of the capture carrier to be mixed can be appropriately adjusted depending on the amount of the target substance contained in the sample.
- This step is a step of adding a dissociator to cleave the bond between avidin and the like in the capture carrier and the biotin derivative (1) to dissociate the avidin and the biotin derivative (1).
- a dissociator In order to mix the mixed solution 1 and the dissociating agent, it is preferable to mix the dissociating agent solution dissolved in the aqueous solvent and the mixed solution 1. After mixing, it is preferable to mix at 10 to 40 ° C. for 1 to 60 minutes. By mixing under such conditions, avidin and the biotin derivative (1) are efficiently dissociated.
- This step may include a step of separating the capture carrier from the liquid mixture 2. When the capture carrier includes magnetic particles, the capture carrier can be easily separated using a magnetic stand.
- Step of separating target substance from liquid mixture 2 This step is a step of separating the target substance contained in the mixed solution 2, and the target substance is separated from the insoluble carrier or the like by this step.
- This step can be carried out by any method, but when the capture carrier contains magnetic particles, the capture carrier can be easily separated using a magnetic stand.
- a method of centrifuging under a centrifugal condition that does not allow the target substance to settle, or a method such as filtering with a filter having a pore size that does not capture the target substance may be used. it can.
- Preparation Example 1 Preparation of desthiobiotin-binding probe molecule (anti-Ep-CAM antibody) 5 mg of desthiobiotin (manufactured by MP Biomedicals) was dissolved in 0.5 ml of dimethyl sulfoxide. In this solution, N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC hydrochloride) were each 5.36 mg (1.2% with respect to the carboxyl group of desthiobiotin). Equivalent) and reacted at room temperature for 60 minutes.
- NHS N-hydroxysuccinimide
- EDC hydrochloride 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
- Ep-CAM antibody an epithelial specific antigen dissolved in 1 ml of PBS (phosphate buffered saline), was separated from this reaction solution (anti-Ep-CAM antibody: clone Ber-EP4, purchased from Dako International) ) 2 mg, and further reacted at room temperature for 3.5 hours. Unreacted desthiobiotin was removed by ultrafiltration to obtain a desthiobiotin-conjugated anti-Ep-CAM antibody.
- PBS phosphate buffered saline
- Preparation Example 3 Preparation of dissociator (1) (synthesis of bovine serum albumin (BSA) conjugated with biotin) 200 mg of biotin (molecular weight: 244.31) was dissolved in 2.76 ml of dimethyl sulfoxide, and 173 mg of NHS and EDC hydrochloride (1.1 equivalent to the carboxyl group of biotin) was added and reacted at room temperature for 60 minutes. 0.508 ml and 1.27 ml were separated from this reaction solution, and 1 g each of BSA (manufactured by SIGMA) was added to a solution in 50 ml of 10 mM phosphate buffer solution (pH 7.0). Reacted for hours.
- BSA bovine serum albumin
- BSA-Biotin6 6 molecules of biotin-bonded dissociator
- BSA-Biotin10 10 molecules of biotin-bonded dissociator
- the amount of biotin bound to BSA was quantified according to the HABA method (4-hydroxyazobenzene-2-carboxylic acid, NM Green, Methods in Enzymology, vol. 18, p418-424, 1970) by Green et al.
- Preparation Example 4 Preparation of dissociator (2) (synthesis of polyacrylic acid bound with biotin derivative) 0.1 g of polyacrylic acid (PAA, manufactured by Wako Pure Chemical Industries, Ltd.) having a molecular weight of 250,000 is dissolved in 5 ml of 10 mM phosphate buffer solution (pH 7.0), and 133 mg of EDC hydrochloride (based on the carboxyl group of polyacrylic acid) 0.5 equivalent).
- PAA polyacrylic acid
- EDC hydrochloride based on the carboxyl group of polyacrylic acid
- a dimethyl sulfoxide solution (10 mg / ml) of a compound represented by the formula (7) which is a biotin derivative having an amino group (Biotin-PEO-LC-Amine; manufactured by Thermo Fisher Scientific Co., Ltd.) 835 ml or 1.67 ml was added, and further reacted at room temperature for 3.5 hours. Unreacted biotin was removed by ultrafiltration to obtain water-soluble biotin-conjugated PAA. Quantification of biotin bound to PAA was performed according to the HABA method.
- PAA-Biotin15 a dissociation agent to which 15 biotin derivatives are bound on average per PAA molecule
- PAA-Biotin40 a dissociation agent to bind 40 biotin derivatives on average per PAA molecule
- Preparation Example 5 Preparation of target cells Remove the medium from the culture dish in which HT-29 cells (target cells) are cultured, add 1 ml of PBS containing 2 mM EDTA, and hold at 37 ° C for 5 minutes to remove the cells from the culture dish. After peeling, the cells were separated into single cells by pipetting and diluted with PBS to obtain a cell dispersion having a cell concentration of 2.0 ⁇ 10 5 cells / ml.
- HT-29 cells express Ep-CAM antigen.
- the number of cells was determined by measuring the amount of genomic DNA by quantitative PCR.
- Proteinase K proteolytic enzyme
- Example 1 Each 1 mg of the capture carrier obtained in Preparation Example 2 was placed in a test tube and dispersed in 1 ml of PBS containing 0.6% by mass citric acid and 0.5% by mass BSA to obtain a capture carrier solution. To this carrier solution for capture, 50 ⁇ l of the cell dispersion obtained in Preparation Example 5 was added and mixed at 4 ° C. for 30 minutes to bind the carrier for capture and HT-29 cells. The capture carrier was then separated using a magnetic stand and washed three times with PBS to separate target cells bound to the capture carrier from unbound cells and other solute components. When the number of cells bound to the separated capture carrier was measured, it was 8,500 cells, 85% of the cells used in the examples.
- the concentration of biotin in the dissociation solution is a value of mass% of biotin calculated from the concentration of the dissociator and the degree of biotinylation of the dissociator.
- Example 2 to 4 The dissociation efficiency was determined in the same manner as in Example 1 except that the dissociating agent shown in Table 1 was used instead of BSA-Biotin6.
- Example 1 Dissociation efficiency was determined in the same manner as in Example 1 except that a PBS solution of 10 mM biotin was used instead of the PBS solution of 0.2% by mass BSA-Biotin6.
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Abstract
Description
例えば、標的物質と結合性を有する抗体等のプローブ分子にビオチンを予め結合させたビオチン標識プローブ分子と、アビジン等が固定化された不溶性担体とを生理的条件下に混合してABC結合を形成させることにより標的物質の捕捉用担体を調製し、この捕捉用担体を用いて標的物質を捕捉することが可能であり、その後にABC結合又はプローブ分子と標的物質の結合を切断することができれば標的物質を分離することが可能である(特許文献1)。
このため、標的物質を分離し易くするため、アビジン等に対する親和性がビオチンよりも低いビオチン誘導体を用いる方法(特許文献2)、ビオチンに対する親和性がアビジンよりも低いアビジン変異体を用いる方法(特許文献3)が知られている。例えば、ビオチンに替えて、アビジン等に対する親和性がビオチンよりも低いデスチオビオチンなどのビオチン誘導体(特許文献2)を用いて上記捕捉用担体を調製し、標的物質を捕捉した後、大量のビオチンを添加することによって、ABC結合を切断し、標的物質を分離する方法が知られている。この場合、ビオチンは、デスチオビオチンとアビジン等の結合に対する競合阻害剤として機能しているものと考えられている。しかし、ビオチンを競合阻害剤として用いるこの方法では、ABC結合を効率良く切断することは難しく、標的物質を効率良く分離することは困難である。
(a)捕捉用担体と標的物質とを水系溶剤中で混合して混合液1を得る工程、
(b)混合液1とビオチン又はその誘導体(2)が結合した水溶性高分子とを混合して混合液2を得る工程、
(c)混合液2から標的物質を分離する工程を含む、方法を提供するものである。
ただし、捕捉用担体は、下記(A)と(B)との結合物である。
(A)不溶性担体に固定化したアビジン若しくはストレプトアビジン
(B)標的物質に対して特異的親和性を有する物質(以下、「プローブ分子」という。)に固定化した、アビジンに対する親和性がビオチンよりも低いビオチン誘導体(1)
(アビジン等とビオチン誘導体の解離方法)
本発明の一実施形態に係るアビジン等とビオチン誘導体(1)の解離方法は、アビジン-ビオチン複合体と、ビオチン又はその誘導体(2)が結合した水溶性高分子とを混合する工程を有する。本発明の解離方法は、通常、水を主成分とする溶媒中で行われる。その場合、アビジン-ビオチン複合体は当該溶媒に溶解していることが好ましいが、アビジン-ビオチン複合体が不溶性の担体に結合している場合等においてはアビジン-ビオチン複合体はその担体と共に溶媒中に分散されていてもよい。
本発明で使用されるアビジン-ビオチン複合体は、アビジン又はストレプトアビジンと、アビジンに対する親和性がビオチン(下記式(3)で表される化合物)よりも低いビオチン誘導体(3)の結合体である。ここで、アビジン又はストレプトアビジンは、一般に市販されているものを用いることができ、これらは、自然界に存在するものを分離・精製したものでもよく、あるいは、遺伝子工学的技術によって人工的に生産されたものであってもよく、ビオチンに対する結合能を失わない範囲で改変されたアビジン又はストレプトアビジンの誘導体であってもよい。アビジン又はストレプトアビジンの誘導体としては、例えば、スクシニル化等の化学的に修飾したアビジン又はストレプトアビジン、通常は4量体であるアビジン又はストレプトアビジンの単量体等が挙げられる。
このようなビオチン誘導体(1)としては、例えば、デスチオビオチン(下記式(4)で表される化合物)、2-イミノビオチン(下記式(5)で表される化合物)、3,4-ジアミノビオチン(下記式(6)で表される化合物)等のビオチンの環状構造部分においてビオチンと相違する誘導体、及びこれらの化学修飾物等が挙げられる。
本発明で使用されるアビジン-ビオチン複合体は、アビジン等を介して不溶性担体に固定化されていてもよい。アビジン-ビオチン複合体が不溶性担体に固定化されていることにより、アビジン-ビオチン複合体を含む分散液から遠心分離、ろ過等の方法によりアビジン-ビオチン複合体を容易に分離することができる。
アビジン等を固定化するために使用する不溶性担体としては、その形状は特に限定されないが、例えば、有機若しくは無機の粒子、平板状基板、微細流路を有する基板、マイクロウェルプレート等が挙げられる。このうち、有機粒子としては、平均粒子径が0.5~10μmのポリスチレン等を主成分とする有機合成高分子からなる粒子が好ましい。さらに、磁気による分離が可能である点で、粒子は磁性粒子であることが好ましい。
磁性粒子の市販品としては、例えば、Dynabeads M-450 Tosylactivated(Invitrogen社製)や、Magnosphere MS300/Carboxyl(JSR株式会社製)等が挙げられる。磁性粒子は、公知の方法(例えば、特公平05-010808号、又は、特開2007-288133号など参照)に従って製造されることが好ましく、タンパクや細胞などの非特異吸着を低減することができる。
本発明で使用されるアビジン-ビオチン複合体は、下記(A)と(B)との結合物(以下、「捕捉用担体」という。)であってもよい。
(A)不溶性担体に固定化したアビジン若しくはストレプトアビジン
(B)標的物質に対して特異的親和性を有する物質(以下、「プローブ分子」という。)に固定化したビオチン誘導体(1)
プローブ分子に固定化するビオチン誘導体(1)は、アビジンに対する親和性が、アビジン-ビオチン複合体の項に記載したビオチンよりも低いビオチン誘導体(1)である。
プローブ分子にビオチン誘導体(1)を結合する方法としては、特に限定されないが、例えば、上述のアビジン等を担体の表面に固定化する方法と同様の方法を用いることができる。例えば、アミノ基を有するプローブ分子を用いる場合、水溶性カルボジイミドなどの脱水縮合剤の存在下で、ビオチンの分子中のカルボキシル基を、プローブ分子中のアミノ基に反応させてアミド結合を形成することにより、ビオチン結合プローブ分子を得ることができる。
図1に、標的物質と結合した捕捉用担体の概念図を示す。
標的物質としては、特に限定されないが、例えば、細胞(例えば、細菌、真菌、動物又は植物の各種細胞)、ウイルス、タンパク(例えば、各種抗原又は抗体、酵素)、1本鎖若しくは2本鎖のDNA若しくはRNA等の核酸、エストロゲン等のステロイド脂質、糖脂質、多糖等が挙げられる。標的物質である細胞(以下、「標的細胞」という。)の具体例としては、特に限定されないが、例えば、正常細胞(例えば造血幹細胞などの幹細胞、白血球などの血球細胞等)、がん細胞(例えば、乳がんや肺がんなどに由来する末梢血中の循環がん細胞、大腸がんなどに由来する糞便中に存在する剥離がん細胞、子宮がんなどに由来する経血中のがん細胞、尿中に存在する膀胱がんなどに由来するがん細胞等)が挙げられる。本発明の標的物質を分離する方法によって、標的物質の分離を効率よく行うことができる。
本発明のアビジン等とビオチン誘導体(1)の解離方法において、アビジン-ビオチン複合体と、ビオチン又はその誘導体(2)が結合した水溶性高分子(以下、「解離剤」ともいう。)を混合することにより、アビジン等とビオチン誘導体(1)との結合を切断して、アビジン等とビオチン誘導体(1)を解離させることができる。
解離剤において用いることができるビオチン誘導体(2)としては、ビオサイチン(biocytin:ビオチン-ε-N-リジン;下記式(7)で表される化合物)、ビオチン-N-ヒドロキシコハク酸イミドエステル(下記式(8)で表される化合物)、下記式(9)で表される化合物(Biotinyl-3,6,9-trioxaundecanediamine)等のビオチンの環状構造部分についてはビオチンと同一の構造を有する誘導体又はアビジン-ビオチン誘導体の項に記載したビオチンの環状構造部分においてビオチンと相違する誘導体を挙げることができる。これらの中では、ビオチンの環状構造部分についてはビオチンと同一の構造を有する誘導体が、アビジンとの親和性が強いため好ましい。
本発明の標的物質を分離する方法は、(a)捕捉用担体と標的物質とを水系溶剤中で混合して混合液1を得る工程、(b)混合液1とビオチン又はその誘導体(2)が結合した水溶性高分子とを混合して混合液2を得る工程、(c)混合液2から標的物質を分離する工程を含む。ここで、捕捉用担体は、
(A)不溶性担体に固定化したアビジン若しくはストレプトアビジンと(B)標的物質に対して特異的親和性を有する物質(以下、「プローブ分子」という。)に固定化したビオチン誘導体(2)との結合物である。
本工程は、捕捉用担体と標的物質を結合させる工程である。具体的には、捕捉用担体と標的物質とを混合することにより、捕捉用担体に含まれるプローブ分子と標的物質又は標的物質中に含まれるプローブ分子と親和性を有する分子が特異的に結合する。
捕捉用担体及び標的物質については、前述の通りである。水系溶剤としては、pH5~9の水を主たる溶媒として含む溶剤が好ましく、リン酸緩衝液、Tris緩衝液などの緩衝液がさらに好ましい。
捕捉用担体と標的物質を混合するには、水系溶剤に分散した捕捉用担体と、同一又は異なる水系溶剤に溶解又は分散した標的物質とを混合することが好ましい。混合後は、10~40℃で1~60分間混和することが好ましい。このような条件で混和することにより、捕捉用担体と標的物質が効率よく結合する。
混合する捕捉用担体の量は、試料中に含まれる標的物質の量により適宜調節することができる。
本工程は、解離剤を添加して捕捉用担体中のアビジン等とビオチン誘導体(1)との結合を切断し、アビジン等とビオチン誘導体(1)を解離させる工程である。混合液1と解離剤を混合するには、水系溶剤に溶解した解離剤の溶液と混合液1を混合することが好ましい。混合後は、10~40℃で1~60分間混和することが好ましい。このような条件で混和することにより、アビジン等とビオチン誘導体(1)が効率よく解離する。
本工程は、混合液2から捕捉用担体を分離する工程を含んでもよい。捕捉用担体が磁性粒子を含む場合には、磁気スタンドを用いて簡便に捕捉用担体を分離することができる。
本工程は、混合液2に含まれる標的物質を分離する工程であり、本工程により標的物質は不溶性担体等から分離される。本工程は、任意の方法により行うことができるが、捕捉用担体が磁性粒子を含む場合には、磁気スタンドを用いて簡便に捕捉用担体を分離することができる。捕捉用担体が磁性粒子を含まない場合には、標的物質を沈降させない程度の遠心条件で遠心分離する方法や、標的物質を捕捉しない程度の孔径を有するフィルターでろ過する等の方法を用いることができる。
デスチオビオチン(MP Biomedicals社製)5mgを0.5mlのジメチルスルホキシドに溶解させた。この溶液にN-ヒドロキシスクシンイミド(NHS)及び1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC塩酸塩)を各5.36mg(デスチオビオチンのカルボキシル基に対して1.2等量)加え、室温で60分間反応した。この反応液から3μlを分け取り、1mlのPBS(リン酸緩衝生理食塩水)に溶解した上皮特異抗原であるEp-CAMに対する抗体(抗Ep-CAM抗体:clone Ber-EP4、ダコインターナショナル社より購入)2mgに加え、さらに室温で3.5時間反応した。未反応のデスチオビオチンを限外ろ過で除去し、デスチオビオチン結合抗Ep-CAM抗体を得た。
ストレプトアビジン磁性粒子(Invitrogen社 Dynabeads M-280 Streptavidin)1mg及び調製例1で得られたデスチオビオチン結合抗Ep-CAM抗体2μgをテストチューブに取り、PBS中で30分間混合し、ストレプトアビジンとデスチオビオチンを結合させた。次いで、磁気スタンドを使って反応液中から磁気粒子を分離し、0.05%Tween20含有PBSで3回洗浄して未反応のデスチオビオチン結合プローブ分子を除去して、デスチオビオチン結合プローブ分子とストレプトアビジン磁性粒子が結合したアビジン-ビオチン複合体(捕捉用担体)を得た。
ビオチン(分子量:244.31)200mgをジメチルスルホキシド2.76mlに溶解し、NHS及びEDC塩酸塩を173mg(ビオチンのカルボキシル基に対して1.1等量)加えて60分間室温で反応した。この反応液から0.508ml、1.27mlを分け取り、それぞれ1gのBSA(SIGMA社製)を50mlの10mMリン酸緩衝溶液(pH7.0)に溶解した溶液に加え、さらに室温で3.5時間反応した。未反応のビオチンを限外ろ過で除去し、水溶性のビオチン結合BSAを得た。以上により、BSA1分子当たり平均で、それぞれ6分子のビオチンが結合した解離剤(BSA-Biotin6という。)及び10分子のビオチンが結合した解離剤(BSA-Biotin10という。)が得られた。BSAに結合したビオチン量は、グリーンらによるHABA法(4-hydroxyazobenzene-2-carboxylic acid、N.M.Green, Methods in Enzymology, vol.18, p418-424, 1970)に従って定量した。
分子量25万のポリアクリル酸(PAA、和光純薬工業製)0.1gを5mlの10mMリン酸緩衝溶液(pH7.0)に溶解し、EDC塩酸塩を133mg(ポリアクリル酸のカルボキシル基に対して0.5等量)加えた。続いて、アミノ基を有するビオチン誘導体である前記式(7)で表される化合物(Biotin-PEO-LC-Amine;サーモフィッシャーサイエンティフィック社製)のジメチルスルホキシド溶液(10mg/ml)を0.835ml又は1.67ml加え、さらに室温で3.5時間反応した。未反応のビオチンを限外ろ過で除去し、水溶性のビオチン結合PAAを得た。PAAに結合したビオチンの定量をHABA法に従って行った。以上により、PAA1分子当たり平均で15分子のビオチン誘導体が結合した解離剤(PAA-Biotin15という。)、PAA1分子当たり平均で40分子のビオチン誘導体が結合した解離剤(PAA-Biotin40という。)が得られた。
HT-29細胞(標的細胞)を培養している培養皿から培地を取り除き、2mM EDTAを含むPBS1mlを加え、37℃で5分間保持して細胞を培養皿から剥がした後、ピペッティングで単細胞に分離し、PBSで希釈して2.0×105細胞/mlの細胞濃度の細胞分散液を得た。HT-29細胞は、Ep-CAM抗原を発現している。
細胞数は、ゲノムDNA量を定量PCR法により測定して求めた。
(1)捕捉用担体に結合した細胞数の測定
各実施例・比較例で得られた捕捉用担体と標的細胞の結合体の全量に0.8mg/mlProteinase K(タンパク分解酵素;QIAGEN社製)の10mM Tris-HCl緩衝液(pH8.3)溶液を50μl加え、55℃で15分加熱し、DNAを溶出させた。続いて、95℃で20分間加熱してProteinase Kを失活させた。得られたDNA溶液20μlを30μlの定量PCR用カクテル(ロシュ社、FastStart Taq使用)に加えて定量PCRを実施した。検量線には、一定数量の細胞から同法により回収したDNA溶液を用いた。定量PCR用の装置は、Applied Biosystems社7500 Real-Time PCR Systemを使用した。
(2)捕捉用担体から分離した細胞数の測定
捕捉用担体と標的細胞の結合体の全量に替えて、各実施例・比較例で解離剤(比較例においてはビオチン)を用いて得られた解離後の細胞分散液全量250μ中の100μlを用いた他は、捕捉用担体に結合した細胞数の測定と同様にして測定し、解離後の細胞分散液全量中の細胞数を測定した。
アビジン等とビオチン誘導体の解離効率は、下式により求めた。
解離効率(%)=捕捉用担体から分離した細胞数÷捕捉用担体に結合した細胞数×100
調製例2で得られた捕捉用担体各1mg分をテストチューブに取り分け、0.6質量%クエン酸及び0.5質量%BSAを含むPBS1ml分散させて捕捉用担体液を得た。この捕捉用担体液に調製例5で得られた細胞分散液50μlを加え、4℃で30分間混和して、捕捉用担体とHT-29細胞を結合させた。次いで、磁気スタンドを使って捕捉用担体を分離し、PBSで3回洗浄することによって、捕捉用担体に結合した標的細胞を未結合細胞やその他の溶質成分から分離した。分離した捕捉用担体に結合していた細胞数を測定したところ、実施例に供した細胞の85%の8,500細胞であった。
標的細胞に結合した捕捉用担体に、調製例3で得られた0.2質量%BSA-Biotin6のPBS溶液250μlを添加し、室温で20分間穏やかに混和した。その後、磁気スタンドを使って粒子を分離し、上清中の解離したHT-29細胞を回収した。光学顕微鏡により観察したところ、上清中の前記細胞はその形態を保持した状態で存在していた。
標的細胞の解離効率を表1に示す。なお、表1において、「解離溶液中のビオチン濃度」とは、解離剤の濃度と解離剤のビオチン化度から計算されたビオチンの質量%の値である。
BSA-Biotin6に替えて表1に記載の解離剤を用いた他は実施例1と同様にして解離効率を求めた。
0.2質量%BSA-Biotin6のPBS溶液に替えて10mMビオチンのPBS溶液を用いた他は実施例1と同様にして解離効率を求めた。
Claims (11)
- アビジンとアビジンに対する親和性がビオチンよりも低いビオチン誘導体(1)の結合体又はストレプトアビジンと前記ビオチン誘導体(1)の結合体(以下、総称して「アビジン-ビオチン複合体」という。)と、ビオチン又はその誘導体(2)が結合した水溶性高分子(以下、「解離剤」という。)を混合する工程を有する、アビジン又はストレプトアビジンとビオチン誘導体(1)を解離させる方法。
- 前記ビオチン誘導体(1)が、ビオチンの環状構造部分においてビオチンと相違する誘導体である、請求項1に記載の方法。
- 前記ビオチン誘導体(1)が、デスチオビオチン、2-イミノビオチン、3,4-ジアミノビオチンから選択される1種以上である、請求項2に記載の方法。
- 前記解離剤の分子量が、ゲルパーミエーションクロマトグラフィーで測定したポリスチレン換算数平均分子量として1,000~1,000,000である請求項1~3のいずれか一項に記載の方法。
- 前記解離剤が、1分子当たり平均で2~2,000個のビオチン又はその誘導体(2)が結合した水溶性高分子である、請求項1~4のいずれか一項に記載の方法。
- 前記解離剤は、水溶性タンパク、水溶性多糖又は水溶性有機合成高分子のいずれかとビオチン又はその誘導体(2)の結合体である、請求項1~5のいずれか一項に記載の方法。
- 前記ビオチン誘導体(2)が、ビオチンの環状構造部分についてはビオチンと同一の構造を有する誘導体である、請求項1~6のいずれか一項に記載の方法。
- 前記アビジン-ビオチン複合体は、不溶性担体に固定化したアビジン若しくはストレプトアビジンとビオチン誘導体の結合体である、請求項1~7のいずれか一項に記載の方法。
- 標的物質を分離する方法であって、
捕捉用担体と標的物質とを水系溶剤中で混合して混合液1を得る工程、
混合液1とビオチン又はその誘導体(2)が結合した水溶性高分子とを混合して混合液2を得る工程、
混合液2から標的物質を分離する工程を含む、方法。
ただし、捕捉用担体は、下記(A)と(B)との結合物である。
(A)不溶性担体に固定化したアビジン若しくはストレプトアビジン
(B)標的物質に対して特異的親和性を有する物質(以下、「プローブ分子」という。)に固定化した、アビジンに対する親和性がビオチンよりも低いビオチン誘導体(1) - 前記標的物質が細胞である、請求項9に記載の方法。
- ビオチン又はその誘導体(2)が結合した水溶性高分子を含有する、アビジン又はストレプトアビジンとビオチン誘導体(1)を解離するために用いられる解離剤。
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CN102575244B (zh) | 2015-07-22 |
JP5686098B2 (ja) | 2015-03-18 |
US8685655B2 (en) | 2014-04-01 |
EP2479268B1 (en) | 2020-11-04 |
JPWO2011034115A1 (ja) | 2013-02-14 |
US20120171763A1 (en) | 2012-07-05 |
CN102575244A (zh) | 2012-07-11 |
EP2479268A1 (en) | 2012-07-25 |
EP2479268A4 (en) | 2014-03-12 |
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