JP4538586B2 - Method for detecting target antigen - Google Patents

Description

  The present invention relates to a method for detecting a target antigen and a detection kit.

  A method using an antigen-antibody reaction is used as a method for detecting foreign proteins such as in-vivo proteins and infected microorganisms in cells or tissues. In this method, a direct method of detecting an antigen by reacting an antigen with a labeled antibody that recognizes the antigen, and an antibody that recognizes the antigen and the antigen (primary antibody) are reacted, and then the primary antibody is recognized. There is an indirect method in which a labeled antibody (secondary antibody) is reacted to detect an antigen, and the indirect method is generally considered highly sensitive.

  In the post-genomic era, there is an increasing need for the development of a technique capable of highly sensitively detecting the expression of a minute amount of protein in cells or tissues and the minute amount of antigen typified by mad cow disease.

  If the antigen used is the same as the antibody, the detection sensitivity of the antigen is determined by the strength of the signal emitted by one molecule of the labeling substance and the number of labeling substances bound to the antibody. Therefore, in order to detect a trace amount of antigen, it is necessary to bind a labeling substance having a strong signal to the antibody or to bind a large amount of labeling substance to the antibody.

  A radioisotope is exemplified as a labeling substance having a strong signal. However, since a radioisotope generates radiation, it requires special equipment, and lacks versatility due to a short effective period. In addition, when a radioisotope is used, direct observation of a cell specimen or tissue specimen is impossible.

  As one of methods for binding a large amount of labeled compound to an antibody, an immuno-PCR method (Immuno-PCR) (see Non-Patent Document 1) in which PCR (Polymerase Chain Reaction) is applied to an antigen-antibody reaction is known. In this method, an antibody and biotinylated DNA are bound through a chimeric protein of protein A and avidin, then the antigen and the antibody are reacted, and the DNA bound to the antibody is used with one or more sets of primers. The antigen is detected by amplifying the DNA fragment and detecting the amplified DNA fragment by agarose gel electrophoresis. Therefore, this method is very effective when a small amount of antigen present in a cell or tissue is extracted from the cell or tissue and detected in a test tube. However, although this method can reveal the presence of an antigen, it cannot reveal the distribution of the antigen in cells or tissues.

  An improved in situ immuno-PCR method (see Non-Patent Document 2) for applying the immuno PCR method to the detection of antigens in cells or tissues is an in situ PCR method. Since a special device used in (see Non-Patent Document 3) is required, and it is necessary to perform in situ hybridization after amplifying the DNA fragment, the operation is complicated. Moreover, there is a possibility that biotinylated DNA having a length necessary for PCR cannot be sufficiently permeated into cells or tissues. Furthermore, since the DNA fragment amplified by PCR is released from the antigen, the distribution of the antigen in the cells or tissues cannot be accurately clarified.

  As the most practical method for binding a large amount of a labeled compound to an antibody, a tyramide signal sensitization method (see Non-Patent Document 4) is known. This method utilizes the property that a tyramide compound is radicalized by the catalytic action of peroxidase in the presence of hydrogen peroxide and nonspecifically covalently binds to an aromatic compound in the vicinity thereof. After reacting the antigen and the peroxidase-conjugated antibody, the tyramide bound to the labeling substance is accumulated by covalently binding to an aromatic compound such as an amino acid contained in a tissue around the peroxidase or a blocking agent. It is a method of sensitization. In the tyramide signal sensitization method, it is important to radicalize tyramide with peroxidase. Since the lifetime of the radical is short, it is very difficult to control the signal / noise ratio.

On the other hand, as a labeling method for labeling an oligonucleotide, a repetitive sequence represented by (XY) _n on the 3 ′ end side (where X and Y comprise a combination of adenine and thymine or uracil, or guanine and cytosine, a method in which an oligonucleotide having n represents an integer of 1 or more is added and extended using a labeled body of a nucleotide constituting the repetitive sequence and a DNA polymerase lacking 5 ′ → 3 ′ exonuclease activity Is known (Patent Document 1). This method is a method of labeling an oligonucleotide by incorporating a labeled nucleotide into an extended portion generated by self-extension of an oligonucleotide having a repeating sequence represented by (XY) _n on the 3 ′ end side.
Japanese Patent Laid-Open No. 11-75880 Sano, T .; Et al., “Science”, 1992, 258, p. 120-122 Cao, P.A. Et al., “The Lancet”, 2000, vol. 356, p. 1002-1003 Nuovo, GJ. , “PCR Methods Appl.”, 1995, Vol. 4, S151-S167. Snappe, PM. Et al., “Journal of Virological Methods”, 1997, Vol. 67, p. 103-112

  An object of this invention is to provide the method and kit which can detect a target antigen with high sensitivity.

  In addition, the present invention provides a method and kit capable of detecting a target antigen with high sensitivity and detecting the intracellular distribution of the target antigen when the target antigen is a cell surface antigen or an intracellular antigen. The purpose is to do.

  In order to achieve the above object, the present invention provides the following methods and kits.

［式中、Ｘはアデニン、チミン又はウラシルを表し、Ｘがアデニンを表すときＹはチミン又はウラシルを表し、Ｘがチミン又はウラシルを表すときＹはアデニンを表し、Ｚは任意の塩基配列を表し、ｎは２以上の整数を表す。］
のいずれかで表される塩基配列からなる一本鎖核酸の５’末端を、前記抗体に結合させる工程
（ｃ）前記工程（ａ）及び（ｂ）を行った後に、デオキシアデノシン三リン酸とデオキシチミジン三リン酸及び／又はデオキシウリジン三リン酸とを含むデオキシヌクレオシド三リン酸の存在下、かつ前記抗原抗体複合体の形成を維持できる温度下において、前記一本鎖核酸にＤＮＡポリメラーゼを作用させる工程
（ｄ）前記工程（ｃ）を行った後に、前記抗原抗体複合体を構成する前記抗体に結合した前記一本鎖核酸の自己伸長を検出する工程 (1) A method for detecting a cell surface antigen or intracellular antigen as a target antigen , comprising the following steps (a) to (d).
(A) reacting the target antigen with one or more antibodies to form an antigen-antibody complex containing the target antigen and the antibody (b) before performing the step (a) Or after having been carried out, the general formulas (I) to (IV):

[Wherein, X represents adenine, thymine or uracil, Y represents thymine or uracil when X represents adenine, Y represents adenine when X represents thymine or uracil, and Z represents an arbitrary base sequence] , N represents an integer of 2 or more. ]
A step of binding the 5 ′ end of a single-stranded nucleic acid comprising the base sequence represented by any of the above to the antibody (c) after performing the steps (a) and (b), deoxyadenosine triphosphate and DNA polymerase acts on the single-stranded nucleic acid in the presence of deoxynucleoside triphosphate containing deoxythymidine triphosphate and / or deoxyuridine triphosphate and at a temperature capable of maintaining the formation of the antigen-antibody complex. (D) a step of detecting self-extension of the single-stranded nucleic acid bound to the antibody constituting the antigen-antibody complex after performing the step (c)

(2) The DNA polymerase used in the step (c) is selected from the group consisting of Escherichia, Bacillus, Thermococcus, Thermus, Pyrococcus, and Thermotoga (Thermotoga) The method according to (1) above, which is a DNA polymerase derived from a microorganism of the genus or a DNA polymerase derived from a bacteriophage.

(3) The said (1) description whose said DNA polymerase used at the said process (c) is a DNA polymerase which does not have one or both of 5 '-> 3' exonuclease activity and 3 '-> 5' exonuclease activity. the method of.

(4) The deoxynucleoside triphosphate used in the step (c) includes labeled deoxyadenosine triphosphate, labeled deoxythymidine triphosphate or labeled deoxyuridine triphosphate,
In the step (d), by detecting the labeled deoxyadenosine triphosphate, the labeled deoxythymidine triphosphate or the labeled deoxyuridine triphosphate incorporated into the extended portion of the single-stranded nucleic acid The method according to (1), wherein self-extension of the single-stranded nucleic acid is detected.

(5) In the step (d), the labeling substance capable of intercalating with the double helix structure of the nucleic acid is reacted with the single stranded nucleic acid to intercalate into the intramolecular double helix structure of the single stranded nucleic acid. The method according to (1), wherein self-extension of the single-stranded nucleic acid is detected by detecting the labeled substance.

(6) The method according to (1), wherein, in the step (b), the 5 ′ end of the single-stranded nucleic acid is bound to the antibody via a bond between avidin or a derivative thereof and biotin or a derivative thereof. .

(7) In the step (b), the 5 ′ end of the single-stranded nucleic acid and the antibody are bound via binding of protein A, protein G or a derivative thereof and the antibody (1) The method described.

(8) The method according to (1) above, wherein in the step (b), the 5 ′ end of the single-stranded nucleic acid and the antibody are bound via a covalent bond.

［式中、Ｘはアデニン、チミン又はウラシルを表し、Ｘがアデニンを表すときＹはチミン又はウラシルを表し、Ｘがチミン又はウラシルを表すときＹはアデニンを表し、Ｚは任意の塩基配列を表し、ｎは２以上の整数を表す。］
のいずれかで表される塩基配列からなる一本鎖核酸とを含む、前記（１）〜（８）のいずれかに記載の標的抗原の検出方法に使用するための標的抗原の検出用キット。 (9) Antibodies against cell surface antigens or intracellular antigens that are target antigens , and general formulas (I) to (IV):

[Wherein, X represents adenine, thymine or uracil, Y represents thymine or uracil when X represents adenine, Y represents adenine when X represents thymine or uracil, and Z represents an arbitrary base sequence] , N represents an integer of 2 or more. ]
A kit for detecting a target antigen for use in the method for detecting a target antigen according to any one of (1) to (8) , comprising a single-stranded nucleic acid having a base sequence represented by any of the above .

  According to the method and kit of the present invention, a target antigen can be detected with high sensitivity. Further, when the target antigen is a cell surface antigen or an intracellular antigen, the target antigen can be detected with high sensitivity, and the intracellular distribution of the target antigen can be detected.

  According to the method and kit of the present invention, the detection sensitivity of an antigen can be freely adjusted by adjusting the degree of self-extension of a single-stranded nucleic acid.

  According to the method and kit of the present invention, the presence or absence of a target antigen can be simultaneously detected for multiple specimens.

The method for detecting a target antigen according to the present invention can include other steps as long as it includes steps (a) to (d).
Hereinafter, steps (a) to (d) will be described in detail.

[Step (a)]
Step (a) is a step of reacting the target antigen with one or more antibodies to form an antigen-antibody complex containing the target antigen and the antibody.

  The target antigen is not particularly limited as long as it can cause an antigen-antibody reaction, and may be an immunogenic antigen (complete antigen), or an immunogenic antigen (incomplete antigen). ). Examples of the target antigen include proteins, peptides, hormones, sugar chains, nucleic acids, lipids and the like. Specifically, among proteins, peptides, hormones, sugar chains, nucleic acids, lipids and the like extracted from cells, blood, urine and the like, a specific one can be selected as a target antigen. In addition, among proteins, peptides, hormones, sugar chains, nucleic acids, lipids and the like extracted from various environmental soils, water, and the like, specific ones can be selected as the target antigen. Furthermore, among proteins, peptides, hormones, sugar chains, nucleic acids, lipids and the like contained on the surface or inside of cells, a specific one can be selected as a target antigen.

  According to the present invention, the presence or absence of a target antigen can be detected with high sensitivity regardless of the type of the target antigen. When the target antigen is a cell surface antigen or intracellular antigen, the presence or absence of the cell surface antigen or intracellular antigen can be detected with high sensitivity, and the intracellular distribution of the cell surface antigen or intracellular antigen can be detected. it can. The cell surface antigen or intracellular antigen is an antigen contained on the surface or inside of a cell that does not constitute a living body (for example, a cell collected from mammals including humans, insects, plants, etc. or cultured cells thereof). .

  The cell surface antigen is not particularly limited, and specific examples thereof include CD antigen, virus receptor, hormone receptor, cell adhesion molecule and the like. Intracellular antigens are not particularly limited, and specific examples thereof include mitochondrial constituent proteins, Golgi constituent proteins, nuclear constituent proteins, and viral antigens.

  As the antibody to be reacted with the target antigen, at least one or more types of antibodies against the target antigen are used. In addition, one or more types of secondary antibodies against the primary antibody, ... , One or more (k + 1) -order antibodies against the k-order antibody can be used. That is, an antigen-antibody complex formed by an antigen-antibody reaction between a target antigen and an antibody includes at least a target antigen and a primary antibody against the target antigen. In addition to this, a secondary antibody against the primary antibody, ..., (k + 1) th antibody against kth antibody can be included. K represents an integer of 1 or more.

  As an antibody to be reacted with a target antigen, usually, one or more primary antibodies against the target antigen are used alone, or one or more primary antibodies against the target antigen and one kind against the primary antibody. Alternatively, two or more secondary antibodies are used in combination. From the viewpoint of improving the detection sensitivity of the target antigen, it is preferable to use one or more primary antibodies against the target antigen in combination with one or more secondary antibodies against the primary antibody.

The antibody may be an antibody fragment as long as it has the desired reactivity (for example, reactivity to a target antigen, reactivity to a primary antibody). Examples of antibody fragments include Fab fragments and F (ab) ' ₂ fragment, single chain antibody (scFv) and the like. The antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody. The monoclonal antibody and the polyclonal antibody may be of any class.

  The antibody can be prepared according to a conventional method. Commercially available antibodies can also be used.

  The antigen-antibody reaction can be performed according to a conventional method, and the reaction conditions are not particularly limited. The antigen-antibody reaction can be performed, for example, according to the following reaction conditions.

  The reaction temperature is usually 4 to 50 ° C, preferably 4 to 42 ° C, more preferably 20 to 40 ° C.

  The reaction time is usually 5 minutes to 24 hours, preferably 10 minutes to 16 hours, and more preferably 30 minutes to 120 minutes.

  As the reaction solvent, for example, a phosphate buffer solution, a carbonate buffer solution, a Tris buffer solution, a tricine buffer solution and the like can be used.

  The amount of the antibody to be reacted with the target antigen is usually 3 to 100 mol, preferably 5 to 50 mol, more preferably 5 to 20 mol, relative to 1 mol of the target antigen.

  When the target antigen and the antibody are reacted, impurities may be present as long as the antigen-antibody reaction is not hindered.

  The antigen-antibody reaction is usually performed on a solid phase such as plastic, glass, nylon membrane, cellulose membrane.

  Prior to the antigen-antibody reaction, the target antigen or antibody may be bound to a solid phase in advance. Thereby, an antibody that does not form an antigen-antibody complex (hereinafter sometimes referred to as “unreacted antibody”) can be easily and efficiently removed. The removal of the unreacted antibody is preferably performed at any time after the step (a) is performed and before the step (d) is performed, and the step (b) is performed after the step (a) is performed. More preferably, it is carried out at any point before performing. By removing the unreacted antibody at any time after performing step (a) and before performing step (b), binding between the unreacted antibody and the single-stranded nucleic acid can be prevented. .

  Examples of the solid phase on which the target antigen or antibody can be immobilized include plastic, glass, nylon membrane, cellulose membrane and the like.

  The target antigen or antibody can be fixed to the solid phase according to a conventional method, and can be fixed through, for example, ionic bond, covalent bond or the like.

  The solid phase washing solution is not particularly limited as long as the formation of the antigen-antibody complex can be maintained. Specific examples thereof include Tween 20-containing phosphate buffered saline, Tween 20-containing Tris buffered saline, and the like. Can be mentioned.

  When the target antigen is a cell surface antigen or intracellular antigen, the cell permeability of the antibody is improved by degreasing the cell membrane by treating the cell with a surfactant such as Triton X-100 before performing the antigen-antibody reaction. It is preferable to make it. Further, when the cells are paraffin-encapsulated cells, it is preferable to expose the antigen by trypsin or protease treatment or microwave treatment.

［式中、Ｘはアデニン、チミン又はウラシルを表し、Ｘがアデニンを表すときＹはチミン又はウラシルを表し、Ｘがチミン又はウラシルを表すときＹはアデニンを表し、Ｚは任意の塩基配列を表し、ｎは２以上の整数を表す。］
のいずれかで表される塩基配列からなる一本鎖核酸の５’末端を、前記抗体に結合させる工程である。[Step (b)]
The step (b) is performed before, during or after the step (a), and the general formulas (I) to (IV):

[Wherein, X represents adenine, thymine or uracil, Y represents thymine or uracil when X represents adenine, Y represents adenine when X represents thymine or uracil, and Z represents an arbitrary base sequence] , N represents an integer of 2 or more. ]
In this step, the 5 ′ end of a single-stranded nucleic acid comprising the base sequence represented by any of the above is bound to the antibody.

  In the general formulas (I) to (IV), “5 ′” represents the 5 ′ end of the single-stranded nucleic acid, and “3 ′” represents the 3 ′ end of the single-stranded nucleic acid.

  In general formulas (I) to (IV), the integer represented by n is 2 or more, preferably 4 or more, and more preferably 7 or more. As the integer represented by n increases, the self-extension of the single-stranded nucleic acid comprising the base sequence represented by the general formulas (I) to (IV) can be efficiently started.

  In general formulas (I) to (IV), the upper limit of the integer represented by n is not particularly limited, but is usually 50, preferably 25, and more preferably 15. Considering that the larger the integer represented by n, the higher the cost required for the synthesis of the single-stranded nucleic acid and the lower the yield, and the larger the integer represented by n, the worse the cell permeability. The upper limit of the integer represented by n is preferably set in the above range.

  In the general formula (III) or (IV), the base sequence represented by Z and the number of bases thereof are not particularly limited. The number of bases of the base sequence represented by Z is usually 2 to 50 bases, preferably 5 to 30 bases, more preferably 10 to 25 bases. In the present specification, the term “base sequence” is used in the sense of including one consisting of two or more bases and one base.

Self-extension of a single-stranded nucleic acid represented by any one of the general formulas (I) to (IV) proceeds as follows. In the following description, a repeated sequence of adenine and thymine and / or uracil is referred to as an “XY sequence”. Examples of the XY sequence include 5 ′-(XY) _t −3 ′, 5 ′-(YX) _t − 3 ', 5'-(XY) _t- X-3 ', 5'-Y- (XY) _t- 3' and the like are included. T represents an integer of 1 or more.

  An XY sequence containing the 3 ′ end of a single-stranded nucleic acid (hereinafter referred to as “3 ′ end XY sequence”) anneals to an XY sequence located 5 ′ of the 3 ′ end XY sequence (intramolecular two by self-annealing). Formation of the main chain). The 3 ′ terminal XY sequence serves as a primer, and the base sequence located on the 5 ′ side of the XY sequence annealed with the 3 ′ terminal XY sequence serves as a template. A complementary base sequence is added to the 3 ′ end of the terminal XY sequence (self-extension by self-priming).

  The longer the base sequence added to the 3 'end of the single-stranded nucleic acid (the extended portion of the single-stranded nucleic acid), the higher the detection sensitivity of self-extension of the single-stranded nucleic acid in step (d). Therefore, it is preferable that the single-stranded nucleic acid can repeat the formation of an intramolecular double strand by self-annealing, self-extension by self-priming, and melting of the intramolecular double strand. In order for a single-stranded nucleic acid to repeat these steps, the base sequence added to the 3 ′ end of the single-stranded nucleic acid is an XY sequence (that is, after the self-extension of the single-stranded nucleic acid, It is necessary that the base sequence is represented by the general formulas (I) to (IV).

  In the case of a single-stranded nucleic acid represented by the general formula (I) or (II), deoxyadenosine triphosphate, deoxythymidine triphosphate and deoxyuridine triphosphate are used as the deoxynucleoside triphosphate used in step (c). The base sequence added to the 3 ′ end of the single-stranded nucleic acid is an XY sequence regardless of whether or not a kind of deoxynucleoside triphosphate other than an acid is contained.

  In the case of a single-stranded nucleic acid represented by the general formula (III) or (IV), the base sequence added to the 3 ′ end of the single-stranded nucleic acid is an XY sequence. The extension reaction using the base sequence represented by Z as a template does not proceed, or the base sequence extending using the base sequence represented by Z as a template must be an XY sequence.

  For example, the deoxynucleoside triphosphate used in step (c) is composed of deoxyadenosine triphosphate and deoxythymidine triphosphate and / or deoxyuridine triphosphate, and includes other types of deoxynucleoside triphosphates If not, if the base sequence represented by Z includes a base sequence consisting only of guanine and / or cytosine on the 3 ′ end side, the base sequence represented by Z from the 3 ′ side of the base sequence represented by Z Since the extension reaction that has proceeded in the direction stops at the 3 ′ end of the base sequence represented by Z, the extension reaction using the base sequence represented by Z as a template does not proceed.

  If the base sequence represented by Z does not contain a base sequence consisting only of guanine and / or cytosine at its 3 ′ end, the extension reaction proceeds to a position containing a base sequence consisting only of guanine and / or cytosine, If the base sequence from the 3 ′ end of the base sequence represented by Z to the position containing the base sequence consisting only of guanine and / or cytosine is an XY sequence, the base extends using the base sequence represented by Z as a template. The sequence is an XY sequence.

  When the base sequence represented by Z includes an XY sequence, the 3 ′ terminal XY sequence may anneal to the base sequence represented by Z, and an extension reaction may proceed using the base sequence represented by Z as a template. . In this case, if the base sequence consisting only of guanine and / or cytosine is adjacent to the 5 ′ end of the XY sequence contained in the base sequence represented by Z, the base extends using the base sequence represented by Z as a template. The sequence is an XY sequence.

  The antibody that binds to the 5 'end of the single-stranded nucleic acid may be any of the antibodies used in step (a). Examples of the antibody that binds the 5 ′ end of the single-stranded nucleic acid include one or more primary antibodies against the target antigen, one or more secondary antibodies against the primary antibody, and the like.

  The method for binding the 5 ′ end of the single-stranded nucleic acid to the antibody is not particularly limited, and specific examples thereof include specific interactions between specific substances, covalent bonds (for example, amide bonds, disulfide bonds, thioethers). And a method using a bond). Appropriate chemical modification can be applied to the antibody and / or nucleic acid according to a conventional method so that the nucleic acid and the antibody can be bound by these methods.

  Specific interaction between specific substances includes, for example, avidin or a derivative thereof (for example, streptavidin, extraavidin, NeutrAvidin) / biotin or a derivative thereof (for example, iminobiotin, carbobiotin, biotin, etc. Hydrazide); IgG Fc fragment / protein A, protein G or derivatives thereof (for example, protein A / G (manufactured by Pierce), which is a fusion protein of protein A and G), except regions that do not contribute to antibody binding Protein G ′ (manufactured by Sigma)); IgG, IgM, IgA, IgE or IgD kappa light chain / protein L or a derivative thereof (for example, protein LA which is a fusion protein of protein L and A (Manufactured by Sigma) Maltose binding protein / maltose; polyhistidine peptide / metal ions such as nickel, cobalt; glutathione-S-transferase / glutathione; calmodulin / calmodulin binding peptide; ATP binding protein / ATP; nucleic acid / complementary nucleic acid; receptor protein / ligand Specific enzyme / substrate interactions, preferred specific interactions include avidin or a derivative thereof / biotin or a derivative thereof; an IgG Fc fragment / protein A, protein G or a derivative thereof specific interaction; Is mentioned.

  When utilizing a specific interaction between the substances a and b, for example, the substance a (for example, biotin or a derivative thereof) is bound to the 5 ′ end of the nucleic acid, and the substance b (for example, avidin or a derivative thereof) is bound to the antigen of the antibody. It binds to a site other than the recognition site. The substance b may be bound to the 5 'end of the nucleic acid, and the substance a may be bound to a site other than the antigen recognition site of the antibody. And a nucleic acid and an antibody can be combined by combining the substance a and the substance b. However, when the antibody itself is substance a or substance b as in the specific interaction of IgG Fc fragment / protein A or protein G, it is not necessary to bind substance a or substance b to the antibody.

  When plural molecules of substance a can bind to one molecule of substance b (for example, when substance a is biotin or a derivative thereof and substance b is avidin or a derivative thereof), substance a is both a nucleic acid and an antibody. Can also be combined. The nucleic acid and the antibody can be bound by binding the substance a bound to the nucleic acid and the substance a bound to the antibody via the substance b.

  Substances that cause specific interactions can be bound to nucleic acids and / or antibodies according to conventional methods.

  For example, by reacting an amino group introduced at the 5 ′ end of a nucleic acid with a carboxyl group of protein A, protein G or a derivative thereof, protein A, protein G or a derivative thereof is converted into a nucleic acid via a peptide bond. Can be combined.

  Alternatively, biotin can be introduced into the 5 'end of the nucleic acid by synthesizing the nucleic acid using biotin amidide.

  In addition, biotin can be bound to the antibody via an amide bond by reacting succinimidyl biotin with the primary amine group of the antibody.

  Further, by reacting the reduced antibody (having a sulfhydryl group) with maleimide-activated avidin, avidin can be bound to the antibody via the bond between maleimide and the sulfhydryl group.

  Further, by reacting an amino group introduced at the 5 'end of a nucleic acid with a carboxyl group possessed by avidin or a derivative thereof, avidin or a derivative thereof can be bound to the nucleic acid via a peptide bond.

  The time point at which the substance that causes the specific interaction is bound to the nucleic acid and / or antibody is not particularly limited as long as it is before the step (b), but the step is performed when or after the step (a) is performed. When performing (b), it is preferable that it is before performing a process (a).

  When utilizing the formation of a covalent bond, the covalent bond can be formed by using a functional group that the nucleic acid or antibody has. Examples of the functional group capable of forming a covalent bond include a carboxyl group, an amino group, a hydroxyl group, a sulfhydryl group, and an aldehyde group.

  Introduction of a desired functional group into a nucleic acid or antibody can be performed using a crosslinking agent capable of reacting with a functional group possessed by the nucleic acid or antibody. Examples of the cross-linking agent include N-succinimidyl (4-iodoacetyl) aminobenzoate (N-succinimidyl (4-iodoacetyl) aminobenzoate) (SIAB), dimaleimide, dithio-bis-nitrobenzoic acid (dithio-bis-). nitrobenzoic acid) (DTNB), N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3- (2-pyridyldithio) propionate (N-succinimidyl) 3- (2-pyridyldithio) propionate) (SPDP), succinimidyl 4- (N-male Domethyl) cyclohexane-1-carboxylate (succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC), 6-hydrazinonicotimide (HYNIC), m-maleimidobenzoyl-N Examples thereof include polyfunctional reagents such as succinimide ester (MBC). (M-aleimidobenzoyl-N-hydroxysuccinimide ester) (MBC)

  For example, when a carboxyl group possessed by an antibody is used, a nucleic acid is activated after activating the carboxyl group with carbodiimides such as 1-ethyl-3- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC). By reacting with the amino group possessed by the antibody, the antibody and the nucleic acid can be amide-bonded. In addition, when an amino group possessed by an antibody is used, the amino group is converted into a carboxyl group using a cyclic acid anhydride such as succinic anhydride, and then reacted with the amino group possessed by the nucleic acid, whereby the antibody and nucleic acid are reacted. And an amide bond.

  The time point at which the step (b) is performed may be any time point before, during or after the step (a), but in the case of utilizing a specific interaction between specific substances, the step When performing (a) or after performing, it is preferable, and when using a covalent bond, it is preferable before performing a process (a).

  If there is a single-stranded nucleic acid that is not bound to an antibody (hereinafter sometimes referred to as “unreacted single-stranded nucleic acid”), or a single-stranded nucleic acid that is bound to an unreacted antibody, one of these nucleic acids. It is preferable to remove the double-stranded nucleic acid before performing the step (d), and it is more preferable to remove the nucleic acid before performing the step (c). By removing before performing step (c), the self-extension of the single-stranded nucleic acid bound to the antibody constituting the antigen-antibody complex can be efficiently initiated and advanced.

[Step (c)]
Step (c) is carried out in the presence of deoxynucleoside triphosphate containing deoxyadenosine triphosphate and deoxythymidine triphosphate and / or deoxyuridine triphosphate after performing steps (a) and (b). And a step of allowing DNA polymerase to act on the single-stranded nucleic acid under a temperature condition capable of maintaining the formation of the antigen-antibody complex.

  As long as deoxynucleoside triphosphates include deoxyadenosine triphosphate and deoxythymidine triphosphate and / or deoxyuridine triphosphate, other types of deoxynucleoside triphosphates (eg, deoxyguanosine triphosphate, deoxy Cytidine triphosphate etc.) may or may not be included.

  The concentration of deoxynucleoside triphosphate is not particularly limited, but is usually 50 μM to 1 mM, preferably 100 to 750 μM, and more preferably 200 to 500 μM.

  The deoxynucleoside triphosphate can comprise labeled deoxyadenosine triphosphate, labeled deoxythymidine triphosphate or labeled deoxyuridine triphosphate. In this case, in step (d), by detecting labeled deoxyadenosine triphosphate, labeled deoxythymidine triphosphate or labeled deoxyuridine triphosphate incorporated into the extended portion of the single-stranded nucleic acid, It is possible to detect self-extension of a double-stranded nucleic acid.

  The mixing ratio of labeled deoxynucleoside triphosphate and unlabeled deoxynucleoside triphosphate is not particularly limited, but is usually 1:10 to 1: 800, preferably 1:20 to 1: 640, more preferably. 1: 40-1: 160.

  The solvent used when the DNA polymerase is allowed to act on the single-stranded nucleic acid is not particularly limited as long as it can maintain the formation of the antigen-antibody complex and does not inhibit the DNA polymerase activity. Examples include acid buffer, Tris buffer, and tricine buffer. These solvents contain a divalent cation necessary for the enzyme reaction, preferably magnesium ion, and may contain serum albumin, glycerol, 2-mercaptoethanol, dithiothreitol and the like as necessary. These substances have an activity protecting action such as oxidation prevention of DNA polymerase.

  The temperature at which the DNA polymerase is allowed to act on the single-stranded nucleic acid is not particularly limited as long as the formation of the antigen-antibody complex can be maintained, but is usually 4 to 75 ° C, preferably 20 to 60 ° C, more preferably 25-45 ° C.

  The time for allowing DNA polymerase to act on the single-stranded nucleic acid is not particularly limited as long as a sufficiently long base sequence can be added to the 3 ′ end of the single-stranded nucleic acid, but usually 30 minutes to 24 hours, preferably Is 1 to 8 hours, more preferably 2 to 5 hours.

  The amount of DNA polymerase that acts on the single-stranded nucleic acid is not particularly limited as long as a sufficiently long base sequence can be added to the 3 ′ end of the single-stranded nucleic acid, but the single-stranded nucleic acid is 1 nmole / 0.1 mL. On the other hand, it is 1-50U normally, Preferably it is 2-25U, More preferably, it is 5-20U.

  The DNA polymerase is not particularly limited as long as the DNA polymerase activity can be exhibited under the temperature condition where the formation of the antigen-antibody complex can be maintained. It is preferable that the DNA polymerase has a DNA polymerase activity capable of adding a sufficiently long base sequence to the 3 'end of the single-stranded nucleic acid under a temperature condition capable of maintaining the formation of the antigen-antibody complex. The term “sufficient length” usually means 1,000 to 50,000 bases, preferably 2,000 to 50,000 bases, more preferably 5,000 to 50,000 bases. It is preferable to have a DNA polymerase activity that can add a base sequence having a length of 30 minutes to 12 hours, preferably 30 minutes to 6 hours, more preferably 30 minutes to 2 hours.

  The DNA polymerase may be naturally occurring as long as it has DNA polymerase activity, or it may be a naturally occurring one modified.

  Examples of DNA polymerases that can exhibit DNA polymerase activity under temperature conditions that can maintain the formation of an antigen-antibody complex include, for example, Escherichia, Bacillus, Thermococcus, Thermus ( Examples include DNA polymerases derived from microorganisms such as Thermus, Pyrococcus, and Thermotoga, and DNA polymerases derived from bacteriophages.

  Examples of the microorganism belonging to the genus Escherichia include Escherichia coli, Escherichia fergusonii, and the like, and the DNA polymerase derived from the microorganism belonging to the genus Escherichia as the microorganism belonging to the genus Escherichia, Examples include polymerase I and Klenow fragment.

  Examples of microorganisms belonging to the genus Bacillus include Bacillus stearothermophilus and Bacillus caldotenax, and DNA polymerase derived from microorganisms belonging to the genus Bacillus Examples thereof include Bst DNA polymerase and BcaBEST DNA polymerase.

  Examples of the microorganisms of the genus Thermococcus include Thermococcus litoralis, Thermococcus 9 ° N-7 (Thermococcus sp. 9 degree N-7), and the like, Thermococcus sp. Examples of the DNA polymerase derived from a microorganism include Tli DNA polymerase, Terminator DNA polymerase, and 9 degree NmDNA polymerase.

  Examples of microorganisms belonging to the genus Thermus include Thermus thermophilus, Thermus aquaticus, Thermus flavus, and the like. Examples of the derived DNA polymerase include Tth DNA polymerase, Taq DNA polymerase, Tfl DNA polymerase, and the like.

  Examples of the microorganism belonging to the genus Pyrococcus include Pyrococcus furiosus, and examples of the DNA polymerase derived from the microorganism belonging to the genus Pyrococcus include Pfu polymerase.

  Examples of microorganisms belonging to the genus Thermotoga include Thermotoga maritima and the like, and examples of DNA polymerases derived from microorganisms belonging to the genus Thermotoga include Ulta polymerase (manufactured by PerkinElmer). Is mentioned.

  Examples of the DNA polymerase derived from bacteriophage include T4 DNA polymerase and T7 DNA polymerase.

  Preferred DNA polymerases include DNA polymerases derived from Escherichia coli, Bacillus stearothermophilus, Thermococcus 9 ° N-7 (Thermococcus sp. 9 degree N-7), and the like. .

  The DNA polymerase preferably does not have one or both of 5 '→ 3' exonuclease activity and 3 '→ 5' exonuclease activity, and more preferably does not have both. There are two reasons for this. One is that DNA polymerases derived from the genus Thermus, Thermococcus litoralis, etc. synthesize DNA even in the absence of templates and primers (Hanaki, K Biochem. Biophys.Res. Commun., 238, 113-118 (1997); Ogata, N. et al., Nucleic Acids Res., 26,4652-4656 (1998)), 5 ′ → 3 ′ exonuclease activity And those modified DNA polymerases lacking 3 ′ → 5 ′ exonuclease activity do not perform DNA synthesis in the absence of template and primers (Hanaki, K. et al., Biochem. Biophys. Res. Comm). n., 244, 210-219 (1998); Hanaki, K. et al., Biochem. Biophys. Res. Commun., 244, 210-219 (1998)), the other being 5 ′ → 3 ′. A DNA polymerase having one or both of an exonuclease activity and a 3 ′ → 5 ′ exonuclease activity digests DNA consisting of a repeat sequence of adenine and thymine (Hanaki, K. et al., Biochem. Biophys. Res. Commun., 244, 210-219 (1998)).

  A DNA polymerase lacking one or both of 5 '→ 3' exonuclease activity and 3 '→ 5' exonuclease activity can be produced by deleting the domain responsible for these activities. Deletion of a predetermined domain can be carried out by artificially introducing mutation using a known method such as site-directed mutagenesis. A commercially available DNA polymerase lacking one or both of 5 '→ 3' exonuclease activity and 3 '→ 5' exonuclease activity can also be used.

  A single-stranded nucleic acid repeats the formation of an intramolecular double strand by self-annealing and the melting of an intramolecular double strand under temperature conditions that can maintain the formation of an antigen-antibody complex. When DNA polymerase acts on, self-extension by self-priming starts and proceeds.

[Step (d)]
Step (d) is a step of detecting self-extension of the single-stranded nucleic acid bound to the antibody constituting the antigen-antibody complex after performing step (c).

  By performing steps (a) to (c), the single-stranded nucleic acid self-extends while the formation of the antigen-antibody complex is maintained. Therefore, in step (d), the antigen-antibody complex can be detected by detecting self-extension of the single-stranded nucleic acid bound to the antibody constituting the antigen-antibody complex. When the target antigen is present, an antigen-antibody complex is formed. When the target antigen is not present, no antigen-antibody complex is formed. Therefore, the target antigen can be detected by detecting the antigen-antibody complex. it can.

  If there is a single-stranded nucleic acid bound to an unreacted antibody or an unreacted single-stranded nucleic acid together with the single-stranded nucleic acid bound to the antibody constituting the antigen-antibody complex, step (d) is performed. It is preferable to remove these single-stranded nucleic acids before. Thereby, the detection accuracy of the self-extension of the single-stranded nucleic acid bound to the antibody constituting the antigen-antibody complex can be improved.

  For a single-stranded nucleic acid bound to an unreacted antibody or an unreacted single-stranded nucleic acid, a target antigen or antibody is immobilized on a solid phase in advance before performing step (a), and then step (d) is performed. It can be removed by washing the solid phase before.

The method for detecting self-extension of a single-stranded nucleic acid is not particularly limited. Self-extension of a single-stranded nucleic acid can be detected, for example, according to the following method.
When the deoxynucleoside triphosphate used in the step (c) includes a labeled deoxyadenosine triphosphate, a labeled deoxythymidine triphosphate or a labeled deoxyuridine triphosphate, a single chain is used in the step (d). Self-extension of single-stranded nucleic acid can be detected by detecting labeled deoxyadenosine triphosphate, labeled deoxythymidine triphosphate or labeled deoxyuridine triphosphate incorporated into the nucleic acid extension. .

  Before detecting the labeled deoxynucleoside triphosphate incorporated into the extended portion of the single-stranded nucleic acid, the labeled deoxynucleoside triphosphate (hereinafter referred to as “unreacted”) that is not incorporated into the extended portion of the single-stranded nucleic acid remains. It is preferable to remove “labeled deoxynucleoside triphosphate”. Thereby, the detection accuracy of the labeled deoxynucleoside triphosphate incorporated into the extended portion of the single-stranded nucleic acid can be improved.

  Unreacted labeled deoxynucleoside triphosphate is removed by preliminarily fixing the target antigen or antibody to the solid phase before performing step (a) and washing the solid phase after performing step (c). can do.

The labeling substance possessed by the labeled deoxynucleoside triphosphate is not particularly limited, and specific examples thereof include fluorescent substances such as fluorescein, rhodamine and phycoerythrin; enzymes such as alkaline phosphatase and horseradish peroxidase; digoxigenin; biotin; Luminol, lucigenin, acridium ester chemiluminescent substances; luciferase, luciferin and other bioluminescent substances; ³² P, ³⁵ S and other isotopes, and the like. Preferred labeling substances include fluorescent dyes such as fluorescein, rhodamine and phycoerythrin; Enzymes such as alkaline phosphatase and horseradish peroxidase; digoxigenin; biotin and the like.

  In addition, in step (d), a labeling substance that can be intercalated into the double helix structure of the nucleic acid is reacted with a single-stranded nucleic acid, and a labeling substance intercalated into the intramolecular double helix structure of the single-stranded nucleic acid is obtained. By detecting, self-extension of a single-stranded nucleic acid can be detected. In addition, since a single-stranded nucleic acid forms an intramolecular double strand by self-annealing, the labeling substance can be intercalated into a single-stranded nucleic acid forming an intramolecular double strand.

  The labeling substance that can intercalate into the double helix structure of the nucleic acid is not particularly limited, and specific examples thereof include ethidium bromide, DAPI (4,6-diamidino-2-phenyllinole), propidium iodide, and the like. A fluorescent substance is mentioned. DAPI is preferable in that it can preferentially intercalate with a base sequence having a large ratio of adenine and thymine.

［式中、Ｘはアデニン、チミン又はウラシルを表し、Ｘがアデニンを表すときＹはチミン又はウラシルを表し、Ｘがチミン又はウラシルを表すときＹはアデニンを表し、Ｚは任意の塩基配列を表し、ｎは２以上の整数を表す。］
のいずれかで表される塩基配列からなる一本鎖核酸とを含む限り、その他の要素を含むことができる。The kit for detecting a target antigen according to the present invention comprises an antibody against the target antigen, and general formulas (I) to (IV):

[Wherein, X represents adenine, thymine or uracil, Y represents thymine or uracil when X represents adenine, Y represents adenine when X represents thymine or uracil, and Z represents an arbitrary base sequence] , N represents an integer of 2 or more. ]
As long as it includes a single-stranded nucleic acid consisting of a base sequence represented by any of the above, other elements can be included.

  Other elements include, for example, deoxynucleoside triphosphates including deoxyadenosine triphosphate and deoxythymidine triphosphate and / or deoxyuridine triphosphate, labeling substance, DNA polymerase, solid phase, reaction solvent, nucleic acid or Examples include reagents for chemically modifying antibodies, and one or more of these can be included.

  The target antigen and the single-stranded nucleic acid comprising the base sequence represented by any one of the general formulas (I) to (IV) may be included in the kit in a bound state, or may not be bound. It may be included in the kit. Further, the kit may be included in the kit in a state that is chemically modified so that it can be bound, or may be included in the kit in a state that is not chemically modified so that it can be bound.

  In the target antigen detection method and detection kit according to the present invention, when the target antigen is a protein, the substance can be specifically bound to the target protein instead of the antibody (hereinafter referred to as “other binding substance”). ) Can be used.

  Examples of the target protein / other binding substance combination include avidin or a derivative thereof (eg, streptavidin, extraavidin, NeutrAvidin) / biotin or a derivative thereof (eg, iminobiotin, carbobiotin) , Biotin hydrazide); antibody or fragment thereof / protein A, protein G, protein L or a derivative thereof (for example, protein A / G (manufactured by Pierce), which is a fusion protein of protein A and G), for binding to an antibody Protein G ′ (manufactured by Sigma) which is a modified form excluding non-contributing regions, protein LA (manufactured by Sigma) which is a fusion protein of protein L and A); receptor protein / ligand (for example, hormone receptor / Environmental Holmo , Fas receptor / Fas ligand); enzyme / substrate; maltose binding protein / maltose; metal ions such as polyhistidine peptide / nickel, cobalt; glutathione-S-transferase / glutathione; calmodulin / calmodulin binding peptide; ATP binding protein / ATP F-actin / farotoxin; deoxyribonuclease / G-actin and the like.

  When other binding substances are used, the single-stranded nucleic acid can be obtained while the formation of the complex containing the target protein and the binding substance is maintained by performing steps (a) to (c). Self-extends. Therefore, in the step (d), the complex can be detected by detecting self-extension of the single-stranded nucleic acid bound to the binding substance constituting the complex. When the target protein is present, the complex is formed, and when the target protein is not present, the complex is not formed. Therefore, the target protein can be detected by detecting the complex.

Hereinafter, the present invention will be described in more detail based on examples.
[Example 1] Preparation of avidinized antibody 0.1 mL of 2 was added to anti-mouse IgG goat IgG (manufactured by Jackson ImmunoResearch) adjusted to a concentration of 5 mg / mL with phosphate buffered saline (PBS, pH 7.6). -Mercaptoethylamine hydrochloride solution (10 g / L 2-Mercaptoethanolamine HCl, 0.1 M Sodium Phosphate, 5 mM EDTA, pH 7.6) was added and treated at 37 ° C for 90 minutes.

  After cooling to room temperature, in order to separate reduced IgG from 2-mercaptoethylamine hydrochloride, 1.1 mL of the reaction solution was passed through a polyacrylamide desalting column, and then a maleimide labeling buffer (0.1 M sodium phosphate) was used. , 5 mM EDTA, pH 7.6).

In order to avidinize the reduced IgG, 1.9 mg / mL reduced IgG and 5 mg / mL maleimide-activated NeutrAvidin Biotin-Binding Protein (Pierce) are mixed in an equal volume of 1 mL, and reacted at room temperature for 3 hours. It was.
In order to remove unreacted avidin and EDTA, the reaction solution is sealed in a dialysis membrane that allows a substance with a molecular weight of 10,000 to pass through, and dialyzed overnight at 4 ° C. using PBS to obtain an avidinized antibody. It was.

[Example 2] Preparation of oligo (dA-dT) -conjugated antibody 0.5 mg of oligo (dA-dT) (5'-atadatadata-3 ') introduced with an amino group at the 5' end was added to 1 mole / L phosphoric acid. After dissolving in 0.5 mL of buffer (pH 7.4), 1 mg of Sulfo-SMCC (Pierce) was added and mixed for 1 hour at room temperature.

  In order to purify the maleimide labeled oligo (dA-dT), the reaction solution was passed through a dextran desalting column, and then the maleimide labeled oligo (dA-dT) was eluted with 1 mL of the maleimide labeling buffer.

  Maleimide-labeled oligo (dA-dT) and 1.9 mg / mL reduced IgG prepared in Example 1 were mixed in an equal volume of 1 mL and reacted at 4 ° C. overnight.

  In order to remove unreacted maleimide-labeled oligo (dA-dT) and EDTA, the reaction solution is sealed in a dialysis membrane that allows a substance having a molecular weight of 10,000 to permeate, and dialyzed overnight at 4 ° C. using PBS. Thus, an oligo (dA-dT) -conjugated antibody was obtained.

[Example 3] Comparison of labeling efficiency by self-extension of oligo (dA-dT) Oligo (dA-dT) (5'-atadataata-3 ') introduced with biotin at the 5' end was converted to TBST (25 mM Tris, pH7). .1 pM, 10 pM and 100 pM oligo (dA-dT) solutions were prepared by dissolving in 2,150 mM NaCl, 0.1% BSA, 0.05% Tween20).

  Each oligo (dA-dT) solution was added dropwise to an avidin-immobilized 96-well plate (Pierce) at 0.1 mL / well (N = 4) and allowed to stand at room temperature for 30 minutes.

  Wells were washed 5 times with TBST to remove unreacted oligo (dA-dT), then 5 U / 100 μL enzyme, 0.2 mM dATP, 0.2 mM dTTP, 2.5 μM digoxigenin labeled dUTP ( An enzyme solution containing dioxygenin-11-dUTP (Roche) was prepared with a buffer attached to each enzyme, and 50 μL / well was added dropwise.

The enzyme used in the enzyme reaction is
(1) Bst polymerase Large fragment (manufactured by NEB, derived from Bacillus steel thermophilus, no 5 ′ → 3 ′ exonuclease activity, no 3 ′ → 5 ′ exonuclease activity) (hereinafter referred to as “Bst-LF”)
(2) ΔTth polymerase (manufactured by Toyobo Co., Ltd., derived from Thermus thermophilus, no 5 ′ → 3 ′ exonuclease activity, no 3 ′ → 5 ′ exonuclease activity)
(3) Klenow fragment (exo-) (manufactured by NEB, derived from Escherichia coli, 5 ′ → 3 ′ exonuclease activity absent, 3 ′ → 5 ′ exonuclease activity absent) (hereinafter referred to as “KF-”)
(4) Klenow fragment (manufactured by Takara Bio Inc., derived from Escherichia coli, without 5 ′ → 3 ′ exonuclease activity, with 3 ′ → 5 ′ exonuclease activity) (hereinafter referred to as “KF”)
(5) KOD polymerase (manufactured by Toyobo Co., Ltd., Thermococcus kodakaraensis, no 5 '→ 3' exonuclease activity, 3 '→ 5' exonuclease activity)
(6) Pfu polymerase (manufactured by Strategene, Pyrococcus furiosus, no 5 '→ 3' exonuclease activity, 3 '→ 5' exonuclease activity)
(7) Stoffel fragment (manufactured by Applied Biosystems, derived from Thermus aquaticus, no 5 ′ → 3 ′ exonuclease activity, no 3 ′ → 5 ′ exonuclease activity) (hereinafter referred to as “SF”)
(8) Therminator polymerase (manufactured by NEB, Thermococcus / 9 ° N-7, 5 ′ → 3 ′ exonuclease not active, 3 ′ → 5 ′ exonuclease not active)
(9) Vent polymerase (manufactured by NEB, derived from Thermococcus litoralis, without 5 ′ → 3 ′ exonuclease activity, with 3 ′ → 5 ′ exonuclease activity).

  Each reaction buffer composition is 20 mM Tris-HCl (pH 8.8), 10 mM potassium chloride, 10 mM ammonium sulfate, 2 mM magnesium sulfide and 0.1% for Bst-LF, Pfu polymerase, Therminator polymerase and Vent polymerase. Triton-based surfactants of ΔTth polymerase and SF, 10 mM Tris-HCl (pH 8.9), 1.5 mM magnesium chloride, 80 mM potassium chloride, 0.1% sodium deoxycholate, 0 1% Triton surfactant and 0.5 mg / mL bovine serum albumin for KF- and KF, 10 mM Tris-HCl (pH 7.5), 5 mM magnesium chloride and 7.5 mM dithiothrei Toll and KOD Polymer For zero, Tris-HCl (pH 8) of 120 mM, a 1.5mM magnesium chloride, 10 mM potassium chloride, ammonium sulfate 6 mM, 0.1% Triton-based surfactant and 10 [mu] g / mL bovine serum albumin.

  After the enzyme reaction was performed at 37 ° C. for 3 hours, the wells were washed 5 times with TBST. 50 μL / well of peroxidase (POD) -labeled anti-digoxigenin Fab fragment (Roche) adjusted to a concentration of 50 mU / mL with TBST was added dropwise at 37 ° C. for 30 minutes, and then the wells were washed 5 times with TBST. The coloring reaction was carried out by dropping a tetramethylbenzidine (Tetramethylbenzidine, Sigma) solution at 30 μL / well in a dark room at room temperature for 10 minutes. After 2N sulfuric acid was added dropwise at 70 μL / well to stop the color reaction, the absorbance at 450 nm was measured with a microplate reader.

  The results are shown in FIG. As shown in FIG. 1, when KF- and Terminator polymerase are used, if the immobilized oligo (dA-dT) is 1 pM or more, the digoxigenin-labeled dUTP incorporated in the extended portion of the oligo (dA-dT) Significantly detected. In addition, when Bst-LF, KF, and Terminator polymerase are used, if the immobilized oligo (dA-dT) is 10 pM or more, the digoxigenin-labeled dUTP incorporated into the extended portion of the oligo (dA-dT) is significantly reduced. It was detected.

[Example 4] Relationship between labeling efficiency due to self-extension of oligo (dA-dT) and labeled dUTP concentration Oligo (dA-dT) (5'-atadataata-3 ') introduced with biotin at the 5' end was measured by TBST. A 10 pM solution was prepared by dissolution, and 0.1 mL / well was added dropwise to an avidin-immobilized 96-well plate. After standing at room temperature for 30 minutes, the wells were washed 5 times with TBST to remove unreacted oligo (dA-dT).

  Next, an enzyme solution containing 5 U / 100 μL of KF- or Terminator polymerase, 0.2 mM dATP, 0.2 mM dTTP, 200 μM, 100 μM, 50 μM, 25 μM, or 12.5 μM digoxigenin-labeled dUTP is added to each enzyme buffer. Prepared and instilled 50 μL / well (N = 4).

For enzyme reaction, the plate was allowed to stand at 37 ° C. or 60 ° C. for 3 hours, and then the wells were washed 5 times with TBST.
Hereinafter, after the same treatment as in Example 3, the absorbance was measured.

  The results are shown in FIG. As shown in FIG. 2, when KF− is 37 ° C., the terminator polymerase is 60 ° C., and the concentration of digoxigenin-labeled dUTP is dTTP: labeled dUTP = 160: 1, dTTP: labeled dUTP = 10: 1 The digoxigenin-labeled dUTP incorporated into the extended part of the oligo (dA-dT) was significantly detected.

[Example 5] Detection of intracellular antigens by self-extension of oligo (dA-dT) at 37 ° C After treatment with 3% formalin for 10 minutes at room temperature, PBS containing 0.5% Triton surfactant was used at room temperature for 5 minutes. Vero cells treated for 1 minute were treated with primary antibody at 37 ° C. for 1 hour and then treated with secondary antibody at 37 ° C. for 30 minutes. Anti-Golgi protein 58K mouse monoclonal antibody (Sigma) diluted 500-fold with PBS as a primary antibody, and biotin-labeled anti-mouse antibody rabbit immune serum diluted 1000-fold with PBS as a secondary antibody (Jackson ImmunoResearch) Used).

  Subsequently, it was treated with streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to a concentration of 2 μg / mL with PBS for 30 minutes at 37 ° C. Finally, biotin was added to the 5 ′ end adjusted to a concentration of 10 nM with PBS. The introduced oligo (dA-dT) (5′-atadataata-3 ′) was treated at 37 ° C. for 30 minutes.

The enzyme used in the enzyme reaction is
(1) Bst-LF
(2) ΔTth polymerase (3) KF-
(4) SF
(5) Therminator polymerase (6) Vent (exo-) polymerase (manufactured by NEB, derived from Thermococcus litoralis, no 5 ′ → 3 ′ exonuclease activity, no 3 ′ → 5 ′ exonuclease activity).

  The reaction buffer composition of Vent (exo-) polymerase is the same as that of Bst-LF.

  The enzyme reaction was performed using a solution containing 5 U / 100 μL of enzyme, 0.2 mM dATP, 0.2 mM dTTP, 0.01 mM rhodamine-labeled dUTP (Tetramethylrhodamine-5-dUTP, manufactured by Roche) in each buffer. And kept at 37 ° C. for 3 hours. Between each treatment, PBS washing by shaking for 5 minutes was performed 3 times. Microscopic observation was performed with an excitation filter: 520 to 550 nm, a fluorescence filter:> 580 nm, and a 60 × oil immersion objective lens.

  The results are shown in FIG. As shown in FIG. 3, when Bst-LF or KF- was used, remarkable staining with rhodamine (red) was observed.

[Example 6] Detection of intracellular antigens by self-extension of oligo (dA-dT) at 60 ° C The same treatment as in Example 5 was conducted except that the enzyme reaction was carried out at 60 ° C for 3 hours.
The results are shown in FIG. As shown in FIG. 4, when Bst-LF, KF-, Therminator polymerase or Vent (exo-) polymerase was used, remarkable staining with rhodamine (red) was observed.

[Example 7] Detection of intracellular antigen by oligo (dA-dT) self-elongation at 74 ° C The same treatment as in Example 5 was conducted except that the enzyme reaction was carried out at 74 ° C for 3 hours.
The results are shown in FIG. As shown in FIG. 5, when Bst-LF or KF- was used, remarkable staining with rhodamine (red) was observed.

[Example 8] Detection of intracellular antigen using DAPI (4,6-diamidino-2-phenylindole) According to the method of Example 5, the enzymatic reaction was carried out using 5 U / 100 μL of KF-, 0.2 mM dATP, 0 This was carried out by incubating at 37 ° C. for 3 hours using a solution containing 2 mM dTTP. The cells were stained with DAPI adjusted to a concentration of 3 nM with PBS together with control cells that had not undergone antigen-antibody reaction. Microscopic observation was performed with an excitation filter: 330-385 nm, a fluorescence filter:> 420 nm, and a 60 × oil immersion objective lens.

  The results are shown in FIG. As shown in FIG. 6, only the nucleus was stained in the control cells, but DAPI-stained nucleic acid was observed in the vicinity of the nucleus in the cells subjected to the oligo (dA-dT) self-extension reaction, which was consistent with the distribution of the Golgi did.

[Example 9] Enzyme reaction time-dependent signal amplification Vero cells treated with 3% formalin for 10 minutes at room temperature and then with PBS containing 0.5% Triton-based surfactant for 10 minutes at room temperature, Treatment with primary antibody at 37 ° C. for 1 hour, followed by treatment with secondary antibody at 37 ° C. for 30 minutes. An anti-GM130 mouse monoclonal antibody (Pharmingen) diluted 4,000 times with PBS containing 0.05% Tween 20 (PBST) was used as the primary antibody, and biotin diluted 500 times with PBST as the secondary antibody. Labeled anti-mouse antibody rabbit immune serum was used.

  Next, treatment with streptavidin adjusted to a concentration of 2 μg / mL with PBST for 30 minutes at room temperature, and finally, oligo (dA-dT) in which biotin was introduced at the 5 ′ end adjusted to a concentration of 1 μM with PBST ( 5′-atatadataata-3 ′) for 30 minutes at room temperature. Between each treatment, PBST washing by shaking for 5 minutes was performed 3 times.

  The enzyme reaction was performed using a solution containing 5 U / 100 μL of KF−, 0.2 mM dATP, 0.2 mM dTTP, 2.5 μM rhodamine-labeled dUTP (10 mM Tris-HCl, pH 7.5, 5 mM magnesium chloride, 7. 5 mM dithiothreitol) and incubated at 37 ° C. for 1, 2 or 4 hours. Microscopic observation was performed with an excitation filter: 520 to 550 nm, a fluorescence filter:> 580 nm, and a 60 × oil immersion objective lens.

  The results are shown in FIG. As shown in FIG. 7, the signal around the cell nucleus (Golgi apparatus) (staining with rhodamine (red)) was enhanced in an enzyme reaction time-dependent manner.

[Example 10] Enzyme reaction time-dependent signal amplification In the same manner as in Example 9, formalin-fixed Vero cells were treated with primary antibody at 37 ° C for 1 hour, and then secondary antibody was used at 37 ° C for 30 minutes. Processed. Anti-ZO-1 mouse monoclonal antibody (manufactured by Pharmingen) diluted 4,000 times with PBST as a primary antibody, and biotin-labeled anti-mouse antibody rabbit immune serum diluted 500 times with PBST as a secondary antibody Using.

  Next, treatment with streptavidin adjusted to a concentration of 2 μg / mL with PBST for 30 minutes at room temperature, and finally, oligo (dA-dT) in which biotin was introduced at the 5 ′ end adjusted to a concentration of 1 μM with PBST ( 5′-atatadataata-3 ′) for 30 minutes at room temperature. Between each treatment, PBST washing by shaking for 5 minutes was performed 3 times.

  The enzyme reaction uses a solution containing 5 U / 100 μL of KF-, 0.2 mM dATP, 0.2 mM dTTP (10 mM Tris-HCl, pH 7.5, 5 mM magnesium chloride, 7.5 mM dithiothreitol). Incubated at 37 ° C. for 1, 2 or 4 hours and washed 3 times with PBST by shaking for 5 minutes. Each cell was stained with DAPI adjusted to a concentration of 1 nM with PBS for 5 minutes at room temperature, and microscopically observed under the same conditions as in Example 8.

  The results are shown in FIG. As shown in FIG. 8, the signal around the cell nucleus (Golgi apparatus) (staining with DAPI) was enhanced in an enzyme reaction time-dependent manner.

[Example 11] Comparison between conventional method and method of the present invention for antigen detection sensitivity Cells were prepared according to the method of Example 5, treated with primary antibody at 37 ° C for 1 hour, and then secondary antibody was used. Treated at 37 ° C. for 30 minutes. Anti-Golgi 58K protein mouse monoclonal antibody (Sigma) diluted 500-fold with PBS as a primary antibody, biotin-labeled anti-mouse antibody rabbit immune serum diluted 200-fold with PBS as a secondary antibody (manufactured by Jackson ImmunoResearch) ) Was used.

  In the conventional method, incubation was performed at 37 ° C. for 30 minutes using fluorescein-labeled streptavidin (manufactured by Sigma) adjusted to a concentration of 2 μg / mL with PBS. In the method of the present invention, in accordance with the method of Example 1, KF- and fluorescein-labeled dUTP were used and incubated at 37 ° C overnight. Microscopic observation was performed with an excitation filter: 470 to 490 nm, a fluorescence filter: 510 to 550 nm, and a 60 × oil immersion objective lens.

  The results are shown in FIG. As shown in FIG. 9, the signal of fluorescein was enhanced in the method of the present invention compared to the conventional method.

[Example 12] Comparison between conventional method and method of the present invention relating to antigen detection sensitivity Cells were prepared according to the method of Example 9, treated with PBS containing 3% hydrogen peroxide at room temperature for 1 hour, and endogenous. Peroxidase was inactivated. Subsequently, it processed at 37 degreeC for 1 hour using the primary antibody. As a primary antibody, an anti-ZO-1 mouse monoclonal antibody prepared by adjusting a 5-fold serial dilution from PBS 500 to 62,500 with PBST was used.

  In the method of the present invention, the treatment was performed under the same conditions as in Example 9 except that fluorescein-labeled dUTP was used instead of rhodamine-labeled dUTP.

  The signal amplification method (TSA) and the indirect antibody method (IF) were selected as conventional methods. In TSA, the reaction was performed according to the package insert of TSA Fluorescence Systems (manufactured by PerkinElmer) except that 500-fold diluted biotin-labeled anti-mouse antibody rabbit immune serum was used as the secondary antibody. For IF, the treatment was performed at 37 ° C. for 1 hour using a FITC-labeled anti-mouse IgG antibody (Sigma) diluted 50 times with PBS.

  The results are shown in FIG. In FIG. 10, (A) is the result of the method of the present invention, (B) is the result of TSA, (C) is the result of IF, and “NC” does not contain the primary antibody (negative control). The results are shown.

  As shown in FIG. 10, the method of the present invention was able to stain the cell membrane site where cells were adhered at a primary antibody concentration 5 times thinner than TSA and 25 times thinner than IF.

[Example 13] Comparison of conventional enzyme immunoassay for antigen detection sensitivity and the method of the present invention A hepatitis B virus surface antigen (HBs) was prepared by dissolving in 50 mM carbonate buffer (pH 9.6) 1, 200 μL of a 2-fold serial dilution series from 024 ng / mL to 1 ng / mL was dropped onto a 96-well immunoplate (Maxi Soap, manufactured by Nargenunk) (N = 3).

  After allowing to stand overnight at 4 ° C., the wells were washed twice with carbonate buffer, and 0.5% BSA-added carbonate buffer was added dropwise at 200 μL / well and allowed to stand at 37 ° C. for 1 hour. After removing the BSA carbonated buffer, 100 μL / well of an anti-HBs mouse monoclonal antibody (Chemicon) diluted 1,000 times with PBST was added dropwise and allowed to stand at 37 ° C. for 30 minutes.

  After the wells were washed three times with PBST, 100 μL / well of a biotin-labeled anti-mouse antibody rabbit immune serum diluted 1,000 times with PBST was added dropwise and incubated at 37 ° C. for 30 minutes. After the wells were washed 3 times with PBST, streptavidin prepared to 500 ng / mL with PBST was added dropwise at 100 μL / well and treated at room temperature for 30 minutes.

  After washing the wells with PBST three times, oligo (dA-dT) (5′-atadataata-3 ′) introduced with biotin at the 5 ′ end adjusted to a concentration of 0.1 μM with PBST was added dropwise at 100 μL / well. Treated for 30 minutes at room temperature. The wells were washed 5 times with PBST, and the wells were further washed twice with PBS.

  Next, an enzyme solution (10 mM Tris-HCl, pH 7.5, 5 mM magnesium chloride, 7.5 mM) containing 5 U / 100 μL of KF−, 0.2 mM dATP, 0.2 mM dTTP, 2.5 μM digoxigenin-labeled dUTP Of dithiothreitol) was added dropwise at 80 μL / well and treated at 37 ° C. for 3 hours.

  After the wells were washed 5 times with PBST, 70 μL / well of a peroxidase (POD) -labeled anti-digoxigenin Fab fragment adjusted to a concentration of 200 mU / mL with PBST was added dropwise and allowed to stand at 37 ° C. for 30 minutes. After the wells were washed 5 times with PBST, a tetramethylbenzidine solution was added dropwise at 50 μL / well, and color development was performed in the dark at room temperature for 5 minutes. 2N sulfuric acid was added dropwise at 100 μL / well to stop the color reaction, and the absorbance at 450 nm was measured with a microplate reader.

  In the conventional method, a peroxidase (POD) -labeled anti-mouse IgG antibody (manufactured by Jackson ImmunoResearch) diluted 500-fold with PBST was used as a secondary antibody, and allowed to stand at 37 ° C. for 30 minutes. After the wells were washed 5 times with PBST, a tetramethylbenzidine solution was added dropwise at 50 μL / well, and color development was performed in the dark at room temperature for 5 minutes. 2N sulfuric acid was added dropwise at 100 μL / well to stop the color reaction, and the absorbance at 450 nm was measured with a microplate reader.

The results are shown in FIG. As shown in FIG. 11, 8 ng / mL HBs could be detected by the method of the present invention, but the conventional method could not be detected unless the HBs was 128 ng / mL or more.
Industrial applicability

  ADVANTAGE OF THE INVENTION According to this invention, the method and kit which can detect a target antigen with high sensitivity using an antigen antibody reaction are provided.

  Further, according to the present invention, when the target antigen is a cell surface antigen or an intracellular antigen, the method and kit can detect the target antigen with high sensitivity and can detect the intracellular distribution of the target antigen. Is provided.

FIG. 1 is a diagram showing a comparison result between various DNA polymerases concerning labeling efficiency by self-extension of oligo (dA-dT).
FIG. 2 is a graph showing the relationship between the labeling efficiency due to self-extension of oligo (dA-dT) and the labeled dUTP concentration.
FIG. 3 is a diagram showing the detection results of intracellular antigens by oligo (dA-dT) self-elongation at 37 ° C.
FIG. 4 is a diagram showing the detection results of intracellular antigens by oligo (dA-dT) self-elongation at 60 ° C.
FIG. 5 shows the detection results of intracellular antigens by self-extension of oligo (dA-dT) at 74 ° C.
FIG. 6 is a diagram showing the detection results of intracellular antigens by nucleic acid staining using DAPI (4,6-diamidino-2-phenylindole).
FIG. 7 is a diagram showing that the signal by the labeling substance (staining with rhodamine (red)) was enhanced in an enzyme reaction time-dependent manner.
FIG. 8 is a graph showing that the signal by the labeling substance (staining with DAPI) was enhanced in an enzyme reaction time-dependent manner.
FIG. 9 is a diagram showing a comparison result between the conventional method and the method of the present invention regarding antigen detection sensitivity.
FIG. 10 is a view showing a comparison result between a conventional method (signal amplification method (TSA) and indirect antibody method (IF)) relating to antigen detection sensitivity and the method of the present invention.
FIG. 11 is a diagram showing a comparison result between a conventional enzyme immunoassay method for antigen detection sensitivity and the method according to the present invention.

Claims (9)

A method for detecting a cell surface antigen or intracellular antigen as a target antigen , comprising the following steps (a) to (d).
(A) reacting the target antigen with one or more antibodies to form an antigen-antibody complex containing the target antigen and the antibody (b) before performing the step (a) Or after having been carried out, the general formulas (I) to (IV):

[Wherein, X represents adenine, thymine or uracil, Y represents thymine or uracil when X represents adenine, Y represents adenine when X represents thymine or uracil, and Z represents an arbitrary base sequence] , N represents an integer of 2 or more. ]
A step of binding the 5 ′ end of a single-stranded nucleic acid comprising the base sequence represented by any of the above to the antibody (c) after performing the steps (a) and (b), deoxyadenosine triphosphate and DNA polymerase acts on the single-stranded nucleic acid in the presence of deoxynucleoside triphosphate containing deoxythymidine triphosphate and / or deoxyuridine triphosphate and at a temperature capable of maintaining the formation of the antigen-antibody complex. (D) a step of detecting self-extension of the single-stranded nucleic acid bound to the antibody constituting the antigen-antibody complex after performing the step (c)   The DNA polymerase used in the step (c) is of the genus Escherichia, Bacillus, Thermococcus, Thermus, Pyrococcus or Thermotoga. The method according to claim 1, which is a DNA polymerase derived from a microorganism or a DNA polymerase derived from a bacteriophage.   The method according to claim 1, wherein the DNA polymerase used in the step (c) is a DNA polymerase that does not have one or both of 5 ′ → 3 ′ exonuclease activity and 3 ′ → 5 ′ exonuclease activity. The deoxynucleoside triphosphate used in step (c) comprises labeled deoxyadenosine triphosphate, labeled deoxythymidine triphosphate or labeled deoxyuridine triphosphate;
In the step (d), by detecting the labeled deoxyadenosine triphosphate, the labeled deoxythymidine triphosphate or the labeled deoxyuridine triphosphate incorporated into the extended portion of the single-stranded nucleic acid The method according to claim 1, wherein self-extension of the single-stranded nucleic acid is detected.   In the step (d), the label that intercalates into the intramolecular double helix structure of the single-stranded nucleic acid by reacting the single-stranded nucleic acid with a labeling substance that can intercalate into the double helix structure of the nucleic acid. The method according to claim 1, wherein self-extension of the single-stranded nucleic acid is detected by detecting a substance.   The method according to claim 1, wherein, in the step (b), the 5 'end of the single-stranded nucleic acid and the antibody are bound via a bond between avidin or a derivative thereof and biotin or a derivative thereof.   The method according to claim 1, wherein in the step (b), the 5 'end of the single-stranded nucleic acid and the antibody are bound via binding of protein A, protein G or a derivative thereof and the antibody.   The method according to claim 1, wherein in step (b), the 5 ′ end of the single-stranded nucleic acid and the antibody are bound via a covalent bond. Antibodies against cell surface antigens or intracellular antigens that are target antigens , and general formulas (I) to (IV):

[Wherein, X represents adenine, thymine or uracil, Y represents thymine or uracil when X represents adenine, Y represents adenine when X represents thymine or uracil, and Z represents an arbitrary base sequence] , N represents an integer of 2 or more. ]
A kit for detecting a target antigen for use in the method for detecting a target antigen according to any one of claims 1 to 8, comprising a single-stranded nucleic acid having a base sequence represented by any of the above .

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