JP2008255046A - Fused protein, carrier for capturing antibody employing the same, and method for detecting antigen using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fused protein that can be adsorbed by a resinous carrier highly densely and highly efficiently, a carrier for capturing an antibody with the fused protein immobilized thereon having excellent capacity of capturing an antibody, and a carrier with an antibody immobilized thereon constituted of the carrier for capturing an antibody with an antibody highly densely integrated thereon. <P>SOLUTION: A fused protein comprising an antibody binding domain and a tag array consisting of 10 to 120 amino acids with the adsorption coefficient of the fused protein with respect to the resinous carrier being greater than 2, a carrier for capturing an antibody with the fused protein immobilized thereon, and a carrier with an antibody immobilized thereon constituted of the carrier for capturing an antibody with an antibody highly densely integrated thereon, are disclosed. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fusion protein with improved adsorption efficiency for a resin, an antibody capture carrier using the same, and an antigen detection method.

  Protein A is a 42 kDa protein produced by Staphylococcus aureus (Staphylococcus aureus bacterium). This protein shows affinity for the constant region (Fc region) of an antibody. Examples of such another protein that binds to an antibody include protein G produced by Streptococcus bacteria and protein L produced by P. streptococcus magnus bacteria. An antibody capture carrier can be prepared by immobilizing these antibody-binding proteins on a suitable carrier. Such antibody capture carriers are used in industrial purification processes for monoclonal antibodies and polyclonal antibodies.

  In addition, antibody-immobilized carriers that capture and detect antigens are known. The antibody-immobilized carrier is used, for example, for antigen detection. Antiwell detection microwell plates on which protein A, protein G, and protein L are immobilized are commercially available. The antibody-immobilized simple substance can be obtained by (1) directly covalently bonding or physically adsorbing the carrier and the antibody, and (2) binding the antibody to the antibody-binding protein immobilized on the carrier. When the method (1) is used, the antibody binds firmly to the carrier. However, in this method, the antibody and the carrier interact directly. For this reason, there is a problem that the antibody is denatured or the orientation of the immobilized antibody is not uniform. On the other hand, when the method (2) is used, antibody denaturation does not occur due to immobilization. In addition, since many antibody binding proteins recognize the constant region of the antibody, the orientation of the immobilized antibody can be controlled. By immobilizing an antibody using an antibody-binding protein in this way, an antibody immobilization carrier having a high specific activity can be obtained. As the antibody capture carrier, for example, the above antibody capture carrier is used.

  In antibody-binding proteins such as protein A, protein G, and protein L, an antibody-binding domain that is an amino acid region having high binding properties to an antibody is specified. For example, protein A has five different antibody binding domains (A to E domains). Protein A is known to be highly recognizable to antibodies derived from mouse, rabid, and human. On the other hand, since it has a plurality of different antibody binding domains, it also binds weakly to antibodies derived from other animal species. Therefore, when used in a sandwich enzyme-linked immunosorbent assay (hereinafter referred to as “ELISA”) using two types of antibodies on a carrier on which an antibody-binding protein such as protein A or protein G is immobilized, the background signal increases. Therefore, it is difficult to detect the antigen with high sensitivity.

  The B domain of protein A is an antibody binding domain consisting of 58 amino acids. This B domain alone exhibits high affinity for antibodies. Further, a Z domain is known in which a mutation is introduced into the B domain to improve stability (see, for example, Non-Patent Document 1). By making this Z domain into a repeating structure, the antibody binding property of the domain is improved. An expression vector for fusing a ZZ domain obtained by repeating the Z domain twice to the end of the target protein has also been designed (see, for example, Non-Patent Document 2). This document discloses that the ZZ domain can be used for purification or extraction of the target protein. In addition, a protein in which a ZZ domain and a green fluorescent protein or a red fluorescent protein are fused has also been created (see, for example, Non-Patent Document 2). This fusion protein can be used as a probe for antigen detection. Therefore, it is useful if the antibody binding domain can be immobilized on a substrate.

  The physical adsorption of the protein to the substrate usually uses the hydrophobic interaction between the protein surface and the substrate. Resin-made carriers on which protein A and protein G are adsorbed using this are already commercially available. On the other hand, generally, as the molecular size of a protein decreases, the hydrophobic interaction tends to decrease and the adsorption efficiency to the substrate tends to decrease. Therefore, it is difficult to efficiently adsorb only the antibody binding domains of protein A and protein G to a resinous carrier.

  In order to solve this problem, for example, a protein in which a fibrous protein is fused to an antibody binding domain terminal in which the C domain of protein G is repeated three times has been developed (see, for example, Patent Document 1). This document discloses that when an antibody binding domain consisting of about 170 amino acids is fused with a fibrous protein consisting of about 360 amino acids, the adsorption to a hydrophobic substrate is improved. Yes.

  In addition, it is disclosed that when a fusion protein of the above fibrous protein and an antibody binding domain is adsorbed to a hydrophobic surface, a structure having irregularities of several nm is formed and adsorbed (for example, non-patent Reference 4).

Furthermore, an example in which a sandwich ELISA was carried out using yeast cells displaying a ZZ domain derived from the B domain of protein A on the cell surface is disclosed (for example, see Non-Patent Document 5).
Bjorn Nilson et al., 8 others, “A synthetic IgG-binding based on staphylococcal protein A”, Protein Engineering, UK, Press Eye Limited (IRL Press Limited), 1987, Vol. 1, No. 2, p. 107-113 Lowennadler, et al., Gene, 1987, 58, p87-97 Qi-Lai Huang, et al., "Application to immunoassays of the fusion protein between protein, ZZ and enchanted protein" Journal of Immunological Methods, ELSEVIER, Netherlands, 2006, volume 309, p. 130-138 Qi-Lai Huang, et al., "Fusion protein between protein ZZ and red fluorescense protein DsRed and samps and caps Biotechnology and Applied Biochemistry, Portland Press Ltd., 2006, Vol. 43, p. 43. Biotechnology and Applied Biochemistry, Portland Press Ltd., UK. 121-127 Gen Tanaka, et al., “Manufacturing of antibody microwell array using self-adhesive antibody-binding protein” (Fabrication of anantibody array with self-binding binding protein biochemistry) Analytical Biochemistry), Elsevier, The Netherlands, 2006, vol. 350, p. 298-303 Wye. Y. Nakamura, et al., “Development of novel whole-cell immunosorbents by the yeast surface fusion display IgG-Y domain surface display of Ig-G domain yeast surface display "Applied Microbiology and Biotechnology, Springer-Verlag, Germany, 2001, vol. 57, p. 500-505 JP-A-2005-220028

  However, in the fusion protein described in Patent Document 1, the antibody binding domain is bound to a fibrous protein of about twice the size. For this reason, the ratio of the antibody binding domain which occupies the whole molecule | numerator becomes small. When such a fusion protein is accumulated on a hydrophobic substrate, the fibrous protein occupies 2/3 of the hydrophobic substrate, while the antibody binding domain occupies 1/3 thereof.

  Further, the uneven structure of the interface described in Non-Patent Document 5 increases the contact efficiency with respect to the antibody and is highly likely to cause non-specific adsorption with respect to proteins other than the antibody.

  Non-Patent Document 6 merely discloses an example in which the detection signal increases when the number of yeast cells is changed. This document does not describe an example of successful application of sensitivity to sandwich ELISA using a conventional microwell plate.

  The present invention has been made in view of the above problems, and an object thereof is to provide a fusion protein that can be adsorbed to a resin carrier with high efficiency and high density.

  Another object of the present invention is to provide an antibody capture carrier having a high antibody capture ability, to which a fusion protein is immobilized. Furthermore, an object of the present invention is to provide an antibody immobilization carrier in which antibodies are integrated at a high density on this antibody capture carrier.

  Still another object of the present invention is to provide an antigen detection method using the above-described antibody-immobilized carrier, particularly an antigen detection method by sandwich ELISA.

  As a result of intensive studies to solve the above problems, the present inventors have created a novel fusion protein and completed the present invention. That is, the present invention is as follows.

  The fusion protein of the present invention is a fusion protein comprising an antibody binding domain and a tag sequence consisting of 10 to 120 amino acids, and the adsorption coefficient of the fusion protein to a resin carrier is larger than 2.

  In the present invention, a tag sequence is added to an antibody binding domain having a small molecular size so as not to exceed the molecular size of the antibody binding domain. This tag sequence contributes to the formation of fusion protein multimers. As a result, the efficiency of adsorption of the fusion protein to the hydrophobic interface of the resin carrier can be promoted.

  In the present invention, the ratio of the antibody binding domain occupying the whole fusion protein is preferably half or more. Also, the molecular size of the fusion protein is small. As a result, when the fusion protein is adsorbed to the resin carrier, the density occupied by the antibody binding domain can be further increased.

式中、Ａは酸性アミノ酸、Ｂは塩基性アミノ酸、Ｘは中性アミノ酸、ｎは２〜２０の整数を示す。 The tag sequence may include a polyion tag containing the same number of acidic amino acids and basic amino acids. The tag sequence may include an expression tag. Alternatively, a polyion tag containing the same number of acidic amino acids and basic amino acids and an expression tag may be used. It is preferable that the polyion tag is a sequence represented by the following general formula (1) or (2).

In the formula, A represents an acidic amino acid, B represents a basic amino acid, X represents a neutral amino acid, and n represents an integer of 2 to 20.

  The tag sequence may be bound to the N-terminus of the antibody binding domain.

  The antibody binding domain may be a part of a sequence constituting protein A or protein G or a repeating structure of a variant thereof. The antibody-binding domain is preferably a protein A B domain or a repeating structure of a B domain variant having the amino acid sequence set forth in SEQ ID NO: 1. It is preferable that the repeating structure of the B domain is repeated twice.

  The antibody capture carrier of the present invention has a resin carrier and the fusion protein immobilized on the resin carrier.

  The antibody capture carrier of the present invention has an antibody binding domain with high antibody specificity in the fusion protein. Further, as described above, the fusion protein can be immobilized on the resin carrier at a high density. As a result, an antibody capture carrier having a high antibody capture ability is obtained.

  The antibody-immobilized carrier of the present invention has a resin carrier, the fusion protein immobilized on the resin carrier, and an antibody bound to the fusion protein. The antibody-immobilized carrier of the present invention can bind an antibody at a high density. As a result, an antibody-immobilized carrier having a high antigen capturing ability can be obtained. The antibody is preferably an antibody derived from an animal species selected from the group of mouse, rabbit, human, guinea pig, dog, cat and pig.

  In the antigen detection method of the present invention, the antigen is detected using an antibody capture carrier in which the fusion protein is immobilized on a resin carrier and an antibody is bound to the fusion protein. The antigen may be detected by a sandwich method. When the antigen detection method of the present invention is used, an antigen can be detected with high sensitivity relative to a conventional sandwich ELISA that does not use an antigen-binding protein. This is a synergistic effect that the fusion protein of the present invention has an excellent adsorptivity to a resin carrier and that the antibody binding domain used in the present invention has high selectivity for antibody species.

  Thus, when the antigen detection method of the present invention is used, trace antigens in urine such as CXCL1 can be detected.

  The fusion protein of the present invention is a fusion protein comprising an antibody binding domain and a tag sequence consisting of 10 to 120 amino acids, and the adsorption coefficient of the fusion protein to a resin carrier is larger than 2. Therefore, it is possible to obtain a resin carrier in which antibody binding domains are integrated at a high density, that is, an antibody capture carrier having a high antibody binding amount. By binding an antibody to this antibody capture carrier, an antibody immobilization carrier in which antibodies are integrated at a high density can be obtained. Further, by using an antibody capture carrier using a fusion protein having an adsorption coefficient of 2, particularly an adsorption coefficient larger than 4, an antigen can be detected with high sensitivity.

  The present invention is described in detail below.

[Fusion protein]
The fusion protein of the present invention is a fusion protein comprising an antibody binding domain and a tag sequence consisting of 10 to 120 amino acids, and the adsorption coefficient of the fusion protein to a resin carrier is larger than 2.

[Antibody binding domain]
The antibody binding domain used in the present invention may be an antibody binding domain derived from protein A or protein G. More preferably, it is a B domain of protein A. As a more preferred form, a Z domain that is a variant of the B domain of protein A is preferred. The amino acid sequence may be further modified while retaining the functional properties of these B domains or the Z domain of SEQ ID NO: 1 shown in Table 1 below. Suitable modifications referred to in the present invention include amino acid modifications for further improving the binding property to antibodies, and amino acid modifications for improving the species selectivity of antibodies. Examples of a method for producing such a variant include random mutation introduction methods such as error-prone PCR and DNA shuffling. Furthermore, as a method for selecting a suitable mutant, various surface display methods such as phage display can be mentioned.

  These antibody binding domains are preferably repetitive sequences. The preferred number of repetitions is preferably 5 times or less since natural protein A is composed of 5 highly homologous domains, and more preferably 2 times.

  The Z domain is composed of 58 amino acids. Therefore, this twice repeated sequence is simply 116 amino acids. However, a peptide sequence having a length that is not functionally impaired and a length may be included as a linker at the linking site of the two domains. This linker sequence is preferably in the range of 1 to 10 amino acids. The linker sequence is not particularly limited.

[Tag array]
The tag sequence in the fusion protein of the present invention is a tag sequence consisting of 10 to 120 amino acids. The fusion protein is physically adsorbed to the resin carrier by this tag sequence. The adsorption coefficient of the fusion protein to the resin carrier is greater than 2. The sequence is not particularly limited as long as the adsorption coefficient of the fusion protein to the resin carrier is larger than 2. The longer the amino acid length, the greater the adsorption coefficient. On the other hand, the longer the amino acid length, the greater the proportion of the tag sequence in the fusion protein. As a result, the number of antibody binding domains occupying per unit surface area is reduced. Further, if the number of amino acids in the tag sequence exceeds 120, it is not desirable because the ratio of non-specific binding of the antibody increases. In the present invention, by binding such a tag sequence to the antibody binding domain, the interaction (electrostatic interaction, hydrophobic binding) between the antibody binding domains is strengthened. As a result, the effect of the present invention can be produced.

  The tag sequence of the present invention includes either a polyion tag, an expression tag, or both a polyion tag and an expression tag. It also includes other functional sequences and any amino acid sequence connecting them. A tag sequence consisting of 10 to 120 amino acids including all of these amino acids may be used.

(Adsorption coefficient)
The adsorption coefficient is the adsorption amount when a 50 μg / mL fusion protein solution is hydrophobically adsorbed on a polystyrene carrier, and only the antibody-binding domain of the same concentration is hydrophobically adsorbed on a polystyrene carrier. It is the value divided by the amount of adsorption. The adsorption coefficient is calculated by the following formula (1).

  The adsorption weight can be calculated by measuring the protein concentration. The adsorption weight is preferably calculated from the decrease rate by calculating the difference between the initial protein solution concentration before adsorption and the protein solution concentration after adsorption. The resin carrier for calculating the adsorption coefficient is preferably a polystyrene resin carrier. The shape of the carrier is preferably a microwell plate.

  As a method for measuring the adsorption weight, the BCA method using bicinchoninic acid (Smith et al., Anal. Biochem. 150.76-85.1985) is suitable.

(Polyion tag)
The tag sequence may include a polyion tag containing the same number of acidic amino acids and basic amino acids. When a polyion tag is used for the tag sequence, a polyion complex is formed. Therefore, even if the number of amino acids constituting the tag sequence is small, the adsorption coefficient of the fusion protein to the resin carrier can be increased.

  As long as the polyion tag includes at least the same number of acidic amino acids and basic amino acids, the polyion tag may have a regular sequence or a random sequence. If the polyion tags are arranged with a certain regularity, electrostatic interaction between the fusion proteins is likely to occur, so that the adsorption coefficient for the resin carrier can be increased.

式中、Ａは酸性アミノ酸、Ｂは塩基性アミノ酸、ｎおよびｘは１〜２０の整数を示す。
これらのポリイオンタグのうち、下記化学式（１）で表される配列が好ましい。

Examples of the polyion tag containing at least the same number of acidic amino acids and basic amino acids include the following sequences. In the formula, A means an acidic amino acid of glutamic acid or aspartic acid, and B means a basic amino acid of lysine or arginine. In the following formulas, the left side of the formula represents the N-terminus of the polypeptide and the right side represents the C-terminus.

In formula, A is an acidic amino acid, B is a basic amino acid, n and x show the integer of 1-20.
Among these polyion tags, a sequence represented by the following chemical formula (1) is preferable.

式中、Ａは酸性アミノ酸、Ｂは塩基性アミノ酸、Ｘは中性アミノ酸、ｎは２〜２０の整数を示す。
これらのポリイオンタグのうち、下記化学式（１）で表される配列が好ましい。

The polyion tag may contain a neutral amino acid in the tag. The neutral amino acid that can be used is not particularly limited as long as it is a neutral amino acid, but is preferably alanine, glycine, leucine, valine, or isoleucine. Examples of the polyion tag including a neutral amino acid in the tag include those having a sequence as shown in the following formula. In the following formula, X represents a neutral amino acid. The neutral amino acids constituting X may be the same or different. When neutral amino acids are contained, acidic amino acids and basic amino acids attract each other due to hydrophobic interaction by neutral amino acids. As a result, the apparent size of the tag sequence in the fusion protein is reduced, so that it can be adsorbed on the resin substrate at high density.

In the formula, A represents an acidic amino acid, B represents a basic amino acid, X represents a neutral amino acid, and n represents an integer of 2 to 20.
Among these polyion tags, a sequence represented by the following chemical formula (1) is preferable.

式中、式中、Ａは酸性アミノ酸、Ｂは塩基性アミノ酸、Ｘは中性アミノ酸、Ｃは、システインを、ｎ及びｍは、それぞれ２〜５の整数を示す。 The polyion tag may be a sequence further containing cysteine in the tag. A polyion complex is usually formed near neutrality. In an acidic or alkaline solution, the charge of the amino acid side chain disappears and a polyion complex cannot be formed. However, if cysteine is included in the polyion tag, a covalent bond (disulfide bond) is formed by sulfur (S) in cysteine. As a result, a stable polyion complex of the fusion protein can be formed even in a solution other than neutral, which is preferable. Examples of the polyion tag containing cysteine in the tag include those having a sequence as shown below.

In the formula, A is an acidic amino acid, B is a basic amino acid, X is a neutral amino acid, C is cysteine, and n and m are each an integer of 2 to 5.

  Thus, when a polyion tag is used, multimers are formed by the formation of ionic bonds or disulfide bonds between molecules. The multimer referred to in the present invention includes a multimer composed of two or more fusion proteins. Thus, in the present invention, the fusion protein can be multiplied by using a polyion tag. By forming a fusion protein multimer, the molecular size increases and the water solubility decreases, so that adsorption to a hydrophobic resin carrier is promoted. Further, the fusion protein multimer may be formed in a solution or on the interface of a resin carrier.

(Expression tag)
The tag sequence of the present invention may be an expression tag used for production of a recombinant protein. The expression tag has functions such as an expression tag for increasing the expression level, an expression tag for affinity purification, an expression tag for antibody detection, and an expression tag for promoting solubilization. As an expression tag that can be used in the present invention, any expression tag may be used as long as it has a peptide sequence in which the total of amino acids of the polyion tag and the expression tag is 10 to 120. Preferred expression tags include His tag, S tag, Myc tag, FLAG tag, T7 tag, HSV tag, perB tag, HA tag, Trx tag, CBP tag, CBD tag, or CBR tag. More preferably, the S tag described in SEQ ID NO: 2 and the His tag described in SEQ ID NO: 3 shown in Table 2 are preferable. These expression tags are not limited to one type, and may be used in combination of two or more types.

  In the tag sequence, the polyion tag and the expression tag may be linked. In the case of linking, the expression tag may be bound to the N-terminal side of the polyion tag.

  The tag sequence may further contain a functional sequence such as a protease recognition sequence such as thrombin or enterokinase.

(Binding of antibody binding domain and fusion protein)
The tag sequence used in the present invention may be bound to either the N-terminus or C-terminus of the antibody binding domain as long as its function is not impaired, but is preferably the N-terminus.

  When the tag sequence is linked to the end of the antibody binding domain, it is preferable to link via a suitable linker sequence. Preferred linker sequences include, but are not limited to, GS linker (Gly-Gly-Gly-Ser) n (n is the number of repetitions). Preferably, a GS linker that is repeated 1 to 3 times (n is an integer of 1 to 3) is used as a linker, and the antibody binding domain and the tag sequence are preferably linked.

[Production of fusion protein]
The fusion protein of the present invention can be produced as a recombinant protein by preparing a gene encoding this amino acid sequence and introducing the gene into a host organism such as E. coli. The host organism used is preferably E. coli. Promoters used for E. coli expression include T7, lac, T7lac, Trp, etc., but are not particularly limited, as long as the promoter can sufficiently express the antibody-binding protein of the present invention. is not.

[Antibody capture carrier]
An antibody capture carrier can be obtained by immobilizing the fusion protein of the present invention on a resin carrier. In the present invention, the antibody capture carrier refers to a carrier capable of selectively binding an antibody.

  The fusion protein is immobilized on the resin carrier by, for example, covalently bonding the fusion protein and the resin carrier using a cross-linking agent or by physically adsorbing the fusion protein and the resin carrier. A preferred method is physical hydrophobic adsorption.

  There is no restriction | limiting in particular as a method of physical adsorption, According to a well-known method. Specifically, the following method is used. Examples include a method in which a resin carrier is immersed in a protein solution of 1 to 1000 μg / mL and incubated for 10 minutes to 48 hours. Further, the fusion protein containing the polyion tag is preferably adsorbed on a resin carrier in a neutral buffer. A suitable neutral buffer is PBS (pH = 7.0). The incubation temperature is preferably 4 ° C to 80 ° C. In particular, the antibody-binding protein having a polyion tag containing a cysteine represented by the above chemical formula 3 is preferably incubated at a temperature higher than room temperature, and more preferably at a temperature of 40 ° C. to 70 ° C. for 3 to 12 hours. A method of incubation is preferred.

  The resin carrier to be immobilized is preferably a highly hydrophobic resin carrier, and suitable resins include polystyrene resin, polymethyl methacrylate resin, latex resin, acrylic resin, polypropylene resin, polyethylene resin, Teflon ( (Registered trademark) resin, ABS resin, nylon resin, epoxy resin, polyurethane resin, alkyd resin, melamine resin, polyimide resin, phenol resin, and the like. In particular, a polystyrene resin, a polymethyl methacrylate resin, and a latex resin are preferable.

  The form of the resin carrier is not particularly limited as long as it is an optimal shape for the application. For example, when used for antibody purification, it is preferably in the form of particles or fibers. Moreover, when using for an antigen detection method, it is preferable that it is plate shape or particle shape, and it is preferable that it is a highly versatile microwell plate shape. More preferably, it is a polystyrene macrowell plate.

  The antibody capture carrier can capture either a monoclonal antibody or a polyclonal antibody. As a preferable antibody for capturing, any antibody derived from mouse, rabbit, human, guinea pig, dog, cat, or pig may be used. More preferred are antibodies produced by these animal species.

  The antibody capture carrier of the present invention can be used as a carrier for separating and purifying the above-mentioned animal-derived antibodies.

  As conditions for capturing (binding) the antibody to the antibody capturing carrier, an antibody solution to be isolated may be brought into contact with the carrier under neutral conditions. In particular, it is preferable to bind the antibody in a buffer solution having a pH of 7.0 to 7.5. A preferred buffer is sodium phosphate buffer.

  The antibody molecules captured by the antibody capture carrier of the present invention can be recovered by setting appropriate elution conditions. As the elution conditions, elution can be performed using an elution buffer having a pH of 3 to 6. The buffer solution is preferably a buffer solution containing sodium citrate.

[Antigen detection]
In the antibody-immobilized carrier of the present invention, a specific antibody against a target antigen molecule is immobilized on an antibody capture carrier. That is, since the antibody-immobilized carrier of the present invention has an antibody immobilized at a high density and the orientation of the antibody is controlled, it can be used effectively in an antigen detection method.

  As an antigen detection method using the above antibody-immobilized carrier, a method of detecting a labeled antigen, a method of capturing an unlabeled antigen on an antibody-immobilized carrier, and detecting using a labeled detection antibody (sandwich method) Two types are possible. In particular, in the sandwich method, unlabeled antigen can be detected, and since the antigen is detected by the specificity of two types of antibodies, it is possible to increase the sensitivity of antigen detection.

  The high sensitivity of antigen detection according to the present invention includes two points: 1) detection signal is improved and calibration curve slope is increased, and 2) (standard deviation of blank signal / calibration curve slope) is reduced. (Based on the definition of the detection limit of the concentration of IUPAC). As the definition of preferable high sensitivity, it is preferable to satisfy the above two.

  The antibody for antigen capture used in the antigen detection method of the present invention is an animal species having specificity for the target antigen molecule and binding to the fusion protein among the antibodies captured by the antibody capture alone. It is preferable that it is an antibody of. Preferred antibody species include any of the antibodies described above, derived from mouse, rabbit, human, guinea pig, dog, cat, and pig. The most preferred antibody is preferably a mouse-derived or rabbit-derived antibody having a high binding property to the ZZ domain.

  The labeled antigen detection antibody used in the antigen detection method of the present invention, particularly the sandwich method, is preferably an antibody derived from an animal species to which the fusion protein does not exhibit affinity. Preferred antibody species are goat-derived and chicken-derived antibodies (polyclonal antibodies). Furthermore, even antibodies other than these animal species can be used as suitable antibodies such as Fab fragments, F (ab ′) 2 fragments (Fab fragment dimers), and single-chain antibodies that do not contain antibody constant regions. It is.

  Examples of the label for the antibody for antigen detection include known labels such as peroxidase label, alkaline phosphatase label, biotin label, fluorescent label, and digoxigenin label. A preferable label for the antibody for antigen detection may be determined based on compatibility with the detection system, and is not limited in the present invention.

  The antigen detection method of the present invention can exert its superiority especially for a peptide antigen having a relatively low molecular weight. In general, for low molecular weight peptide antigens, the detection limit tends to decrease. The low molecular weight peptide antigen referred to in the present invention refers to an antigen of 20 kDa or less. In particular, in the sandwich ELISA, if the molecular size of the antigen is small, even if the antigen is bound to the solidified antibody, it is greatly affected by the orientation. For this reason, an antigen cannot be detected with high sensitivity. By using the antigen detection method of the present invention, the orientation of the solidified antibody is controlled, and the low molecular weight peptide captured by the antibody is also presented on the solution side. Due to these effects, the antigen detection method of the present invention particularly improves the detection sensitivity of low molecular weight peptide antigens.

  The antigen detection method of the present invention can measure antigen molecules derived from human body fluids. Preferred samples include blood, serum, biological tissue extract, urine, stool, saliva and the like.

  Use of the antigen detection method of the present invention makes it possible to detect an antigen with higher sensitivity than conventional antigen detection methods. Even if the measurement sample is urine with a low antigen concentration, the antigen can be detected. The antigen in urine is preferably a protein that becomes a cancer marker, for example, and particularly preferably a renal cancer marker or a bladder cancer marker. More preferably, it is a bladder cancer marker. For example, CXCL1 concentration, which is a protein specific to bladder cancer, can be measured from human urine.

  EXAMPLES Examples will be given below to describe the present invention in detail, but the present invention is not limited to the examples.

  In the following examples, for the amino acids constituting the polyion tag, alphabets of alanine: A, cysteine: C, glutamic acid: E, and lysine: K were used as abbreviations indicating the respective amino acids.

[Preparation of fusion protein expression vector]
(Example)
(Production of antibody binding domain)
As the antibody binding domain, a sequence in which the Z domain derived from the B domain of protein A was repeated twice was used. The Z domain gene was prepared by preparing a sense strand and an antisense strand synthetic oligonucleotide encoding SEQ ID NO: 1 shown in Table 1 and annealing them. The gene encoding the Z domain was cloned into the pT7Blue vector (Novagen) by TA cloning to obtain pT7-Z1. Next, from pT7-Z1, the Z domain was excised with restriction enzymes SpeI and NheI, subjected to agarose gel migration, and then stained to purify the Z domain fragment. The purified Z domain fragment was further cloned into the NheI cleavage site of pT7-Z1 to produce pT7-ZZ.

Example 1
Next, it was cloned into a ZZ domain pET30 (a) vector (Novagen). From pT7-ZZ, the ZZ domain was cleaved with BamHI and SalI, purified by agarose gel electrophoresis. The resulting ZZ domain fragment was cloned into the BamHI and SalI sites of the pET30 (a) vector. Furthermore, oligo DNA having a TAA stop codon was inserted into the SalI site to obtain pET-ZZ. The amino acid sequence of the fusion protein “tag ZZ” (Example 1) encoded by pET-ZZ is shown in SEQ ID NO: 4 in Table 3 below. The fusion protein “tag ZZ” is a fusion protein having a tag sequence that contains an expression tag but does not have a polyion tag. In addition, the tag sequence portion includes a His tag, an S tag, and a prosthesis cleavage sequence, which are expression tags derived from the pET30 (a) vector.

(Example 2)
Oligonucleotides encoding polyion tag 1 ((K-A-E-G) ₂ CG) ₂ ) by heating the oligonucleotides described in Table 4 below and listed in SEQ ID NOs: 6 and 7 to 100 ° C. and then cooling. DNA was prepared. The oligo DNA was cloned into the restriction enzyme NcoI and BamHI sites of pET-ZZ to prepare pET-SELF. The amino acid sequence of the fusion protein “SelfZZ” (Example 2) encoded by pET-SELF is shown in SEQ ID NO: 4 in Table 5. The fusion protein “SelfZZ” of Example 1 is a fusion protein having a tag sequence including a polyion tag.

(Example 3)
Oligonucleotides encoding polyion tag 2 ((K) 7 (E) 7) were prepared by heating the oligonucleotides described in Table 6 below in SEQ ID NOS: 8 and 9 to 100 ° C. and then cooling. The oligo DNA was cloned into the restriction enzyme NcoI and BamHI sites of pET-ZZ to produce pET-K7E7. The amino acid sequence of the fusion protein “K7E7ZZ” (Example 3) encoded by pET-K7E7 is shown in SEQ ID NO: 10 in Table 7. The fusion protein “K7E7ZZ” of Example 1 is a fusion protein having a tag sequence including a polyion tag.

Example 4
Oligonucleotides encoding the comparative tag 1 (K7) were prepared by heating the oligonucleotides described in Table 8 below in SEQ ID NOS: 11 and 12 to 100 ° C. and then cooling. The oligo DNA was cloned into the restriction enzyme NcoI and BamHI sites of pET-ZZ to produce pET-K7. The amino acid sequence of the fusion protein “K7ZZ” (Example 4) encoded by pET-K7 is shown in SEQ ID NO: 13 in Table 9. The fusion protein of Example 4 is a fusion protein containing an expression tag and not containing a polyion tag.

(Fusion protein expression conditions and purification)
The obtained pET-ZZ, pET-SELF, pET-K7E7, and pET-K7 were transformed into E. coli BL21 (DE3) as a host to prepare E. coli for expression. Expression of the fusion protein was performed in LB medium (kanamycin added). These E. coli were cultured with shaking at 37 ° C. until OD 0.6 was reached. Thereafter, IPTG was added to a final concentration of 0.1 mM and cultured for 12 hours. After culturing, E. coli was collected by centrifugation and sonicated in a buffer containing 8M urea. Insoluble residue was removed by centrifugation, and a cell-free extract was prepared. The fusion protein was purified from the cell-free extract using a metal chelate resin.

  The purified fusion protein solution was replaced with PBS (pH = 7.0) using a dialysis membrane having a molecular weight of 10,000 cutoff value. The fusion protein solution obtained as a result of dialysis was colorimetrically determined for protein concentration using a BCA measurement reagent and albumin having a known concentration.

(Comparative Example 1)
The ZZ (control) peptide used as Comparative Example 1 was obtained by treating the tag ZZ of Example 1 obtained by the above fusion protein expression conditions and purification with protease to remove the tag portion. The protease treatment of tag ZZ was performed using enterokinase (Invitrogen). Enterokinase was removed from the solution using EK way resin (Invitrogen). Dialysis and protein concentration measurement were performed in the same procedure as described above.

[Measurement of adsorption amount of fusion protein and calculation of adsorption coefficient]
Using the obtained fusion proteins of Example 1 “Tag ZZ”, Example 2 “Self ZZ”, Example 3 “K7E7ZZ”, Example 4 “K7ZZ” and Comparative Example 1 “ZZ”, the following method was used. Was used to measure the amount of adsorption on the resin carrier, and the adsorption coefficient was determined.

The amount of the fusion protein adsorbed on the polystyrene plate is determined by adding 100 μL of 50 μg / mL (PBS solution) of the fusion protein solution to a 96-well polystyrene plate (manufactured by Nunk, Maxisorp 96-well plate) and incubating at 50 ° C. for 3 hours It was measured by. The amount of fusion protein adsorbed was calculated by the decrease rate of the protein concentration contained in the fusion protein solution before and after incubation. The protein concentration was measured using MicroBCA assay kit (Pierce). The adsorption coefficient of the prepared fusion protein was calculated based on Equation 1 above. Table 10 shows the measured value of the fusion protein adsorption amount and the calculated value of the adsorption coefficient.

  From Table 10, the fusion proteins of Examples 1 to 3 of the present invention have an adsorption coefficient of 2.81, 4.5, and 4.8 for the resin carrier, which is superior to Comparative Example 1 due to the effect of the expression tag. You can see that

  Table 10 also shows that the fusion proteins of Examples 2 to 3 of the present invention have an adsorption coefficient with respect to the resin carrier of greater than 4, and are extremely excellent due to the synergistic effect of the expression tag and the polyion tag. .

[Calculation of antibody binding rate to antibody capture carrier]
Fusion of each fusion protein of Example 1 (tag ZZ), Example 2 (SelfZZ), Example 3 “K7E7ZZ”, Example 4 “K7ZZ” and Comparative Example 1 (ZZ) to a 96-well polystyrene plate at 50 μg / mL 100 μL of protein solution was added and incubated at 50 ° C. for 3 hours for adsorption.

  After incubation, the fusion protein solution was removed, washed 3 times with 250 μL of PBST (Tween 0.05%), and further washed 3 times with PBS (pH 7.0) to prepare an antibody capture plate as an antibody capture carrier. . For control, a plate that did not adsorb the fusion protein was used as an antibody capture carrier for control.

A peroxidase-labeled rabbit IgG antibody (manufactured by Sigma) solution 100 μL (PBS) was added to the antibody capture carrier (antibody capture plate). After adding the antibody solution, it was incubated at room temperature for 6 hours. After incubation, the antibody solution was removed and washed 3 times with PBST (Tween 0.05%). After washing, TMB reagent (100 μL) was added to the plate and incubated at room temperature for 20 minutes. Thereafter, 1N HCl (100 μL) was added to stop the reaction. Absorbance was measured by measuring a wavelength of 450 nm, and the antibody immobilization rate was measured. The antibody immobilization ratio was calculated by taking the absorbance of an untreated plate for control as 1, and calculating the relative value. The results are shown in Table 11.

  From Table 11, the fusion protein of the present invention has an antibody immobilization rate of 16 (Example 1), 23 (Example 2), 25 (Implementation) compared to the antibody immobilization rate of 7.7 of Comparative Example 1 with only the ZZ domain. Example 3) 10 (Example 4) is high.

[Comparison in sandwich ELISA of CXCL1 (GROalpha)]
The CXCL1 sandwich ELISA used a mouse IgG monoclonal antibody as a capture antibody and a biotin-labeled goat polyclonal antibody as a detection antibody. For detection, HRP-labeled avidin was used, and TMB was used as a substrate. The antibody reagent used was manufactured by R & D. Recombinant CXCL1 prepared 300,150,100,50,25,10.5,0pg / mL diluted solution, and used as a standard.

A capture antibody (PBS solution) was added to the antibody capture carrier and incubated for 6 hours. After removing the antibody solution, it was washed 3 times with PBST. 1% BSA (PBS) solution was then added and incubated for 12 hours. After removing the BSA solution, the plate was washed 3 times with PBST to prepare an antigen capture plate. Next, CXCL1 diluted solution (PBST) was added and incubated for 2 hours. After incubation, the CXCL1 solution was removed and washed 3 times with PBST. Next, a biotin-labeled antibody was added and incubated for 2 hours. After incubation, the antibody solution was removed and washed 3 times with PBST. HRP labeled avidin was then added and incubated for 1 hour. Then, it was washed 3 times with PBST. Next, TMB coloring reagent was added and incubated for 20 minutes. Thereafter, 1N HCl was added to stop the reaction. Thereafter, a color development value of 450 nm was measured with a microplate reader. FIG. 1 shows the measurement results. Table 12 shows the regression line slope and contribution rate (IUPAC definition) of each plot in the concentration range 0 to 300 pg / mL calculated from the data of FIG.

  From FIG. 1 and Table 12, it was revealed that when the fusion protein of the example of the present invention was used, the contribution ratio was as large as 0.999, and the ELISA detection signal was greatly improved. In addition, the slope of the calibration curve also increases, indicating that the detection sensitivity has been improved.

  Further, when comparing the tag ZZ (Example 1) and the SelfZZ (Example 2), their adsorption coefficients are greatly different from 2.81 and 4.5, respectively. However, there was no significant difference in detection sensitivity. From this, it was shown that the detection sensitivity of the fusion protein used in the ELISA can be sufficiently improved when the adsorption coefficient is larger than 2.

[Comparison in sandwich ELISA using protein A coated plate]
Using the fusion protein of Example 2 above and a carrier coated with Protein A (Comparative Example 2), a comparison in sandwich ELISA was performed in the same manner as described above. FIG. 2 shows the measurement results. Table 13 shows the regression line slope of each plot in the concentration range of 0 to 100 pg / mL calculated from the data of FIG.

  From the above results, it was shown that when sandwich ELISA was performed with a carrier coated with protein A, the detection signal increased, but the calibration curve slope decreased (FIG. 2). On the other hand, in the case of Example 1 of the present invention, the ELISA detection signal is greatly improved, the slope of the calibration curve is also increased, and the detection sensitivity is improved. That is, in the sandwich ELISA, it was found that the use of the fusion protein using the Z domain, which is an antibody-binding partial sequence derived from protein A, is more suitable for quantification than protein A.

[Comparison of sandwich ELISA with CXCL10]
The CXCL10 sandwich ELISA used a mouse IgG monoclonal antibody as a capture antibody and a biotin-labeled goat polyclonal antibody as a detection antibody. For detection, HRP-labeled avidin was used, and TMB was used as a substrate. Recombinant CXCL10 using the above antibody reagent manufactured by R & D was used to prepare a diluted solution of 250, 125, 63, 31, 15, 0 pg / mL and used as a standard. The conditions were the same as the comparison in the above-mentioned CXCL1 sandwich ELISA. FIG. 3 shows the measurement results. Table 14 shows the regression line slope and contribution rate (IUPAC definition) of each plot in the concentration range 0 to 125 pg / mL calculated from the data of FIG.

From this result, it became clear from FIG. 3 and Table 14 that the ELISA detection signal was greatly improved when the fusion protein of the example of the present invention was used. In addition, the slope of the calibration curve has also increased compared to the comparative example, indicating that the detection sensitivity has been improved. Using the fusion protein of the present invention, it was found that the detection sensitivity was improved even for antigens other than CXCL1.

FIG. 1 shows the ELISA results for CXCL1. The color development values when using 4 types of antibody binding plates (untreated plate, ZZ (control), tag ZZ, SelfZZ) are shown. FIG. 2 shows ELISA results of CXCL1 using protein A coated plates. The color values when using three types of antibody binding plates (untreated plate, protein A coated plate, SelfZZ) are shown. FIG. 3 shows ELISA results for CXCL10. The color development values when using four types of antibody binding plates (untreated plate, ZZ (control), tag ZZ, SelfZZ) are shown.

Claims (16)

A fusion protein comprising an antibody binding domain and a tag sequence consisting of 10 to 120 amino acids, wherein the adsorption coefficient of the fusion protein to a resin carrier is greater than 2.
Fusion protein.   The fusion protein according to claim 1, wherein the tag sequence has a polyion tag containing the same number of acidic amino acids and basic amino acids.   The fusion protein according to claim 1, wherein the tag sequence has an expression tag.   The fusion protein according to claim 1, wherein the tag sequence has a polyion tag containing the same number of acidic amino acids and basic amino acids, and an expression tag. The fusion protein according to claim 2 or 4, wherein the polyion tag has a sequence represented by the following general formula (1) or (2).

In the formula, A represents an acidic amino acid, B represents a basic amino acid, X represents a neutral amino acid, and n represents an integer of 2 to 20.   The fusion protein according to any one of claims 1 to 5, wherein the tag sequence is bound to the N-terminus of the antibody binding domain.   The fusion protein according to any one of claims 1 to 6, wherein the antibody-binding domain is a part of a sequence constituting protein A or protein G or a repeating structure thereof.   The fusion protein according to claim 7, wherein the antibody-binding domain is a B domain of protein A or a variant of the B domain having the amino acid sequence set forth in SEQ ID NO: 1.   The fusion protein according to claim 8, wherein the antibody-binding domain is a repeating structure of a variant of a B domain having the amino acid sequence set forth in SEQ ID NO: 1. A resin carrier;
The fusion protein according to any one of claims 1 to 9, which is immobilized on a resin carrier,
An antibody capture carrier having A resin carrier;
The fusion protein according to any one of claims 1 to 9, which is immobilized on a resin carrier,
An antibody-immobilized carrier having an antibody bound to the fusion protein.   The antibody-immobilized carrier according to claim 11, wherein the antibody is an antibody derived from an animal species selected from the group consisting of mouse, rabbit, human, guinea pig, dog, cat and pig. Immobilizing the fusion protein according to any one of claims 1 to 9 on a resin carrier,
Using an antibody capture carrier in which an antibody is bound to this fusion protein,
An antigen detection method for detecting an antigen.   The antigen detection method according to claim 13, wherein the detection of the antigen is a sandwich method.   The antigen detection method according to claim 14, wherein the antibody for antigen detection used in the sandwich method is any antibody selected from the group consisting of a goat-derived polyclonal antibody, a chicken-derived polyclonal antibody, a Fab fragment, and a single-chain antibody. The method for detecting an antigen according to claim 14 or 15, wherein an antigen in urine is detected.

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