WO2014192202A1 - Protéine recombinée capable de se lier de façon spécifique et rapide à la troponine i du tissu myocardique humain - Google Patents

Protéine recombinée capable de se lier de façon spécifique et rapide à la troponine i du tissu myocardique humain Download PDF

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WO2014192202A1
WO2014192202A1 PCT/JP2014/001219 JP2014001219W WO2014192202A1 WO 2014192202 A1 WO2014192202 A1 WO 2014192202A1 JP 2014001219 W JP2014001219 W JP 2014001219W WO 2014192202 A1 WO2014192202 A1 WO 2014192202A1
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recombinant protein
seq
antibody fragment
troponin
variable region
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PCT/JP2014/001219
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English (en)
Japanese (ja)
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淳 福永
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パナソニックヘルスケア株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to a recombinant protein capable of specifically and rapidly binding to human cardiac muscle-derived troponin I.
  • Non-Patent Document 1 and Non-Patent Document 2 disclose that the concentration of myocardial troponin I rapidly increases in the blood of patients suffering from acute myocardial infarction.
  • An object of the present invention is to provide a recombinant protein capable of specifically and rapidly binding to human cardiac muscle-derived troponin I.
  • a recombinant protein that specifically binds to troponin I from human myocardium A recombinant protein comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 63; and a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 65.
  • the recombinant protein according to item A1, wherein the recombinant protein is an antibody fragment.
  • A6 The recombinant protein according to item A3, wherein the antibody fragment is an scFv antibody fragment.
  • B1. A method for binding a recombinant protein to troponin I derived from human cardiac muscle, Preparing the recombinant protein (a); wherein the recombinant protein comprises: A light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 63; and a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 65; And contacting the recombinant protein with troponin I derived from human myocardium and binding the recombinant protein to troponin I derived from human myocardium (b) Including the method.
  • B2 The method according to item B1, wherein the recombinant protein is an antibody.
  • B3. The method according to item B1, wherein the recombinant protein is an antibody fragment.
  • B4. The method according to item B3, wherein the antibody fragment is a Fab antibody fragment.
  • B5. The method of item B3, wherein the antibody fragment is an F (ab ′) 2 antibody fragment.
  • B6. The method according to item B3, wherein the antibody fragment is a scFv antibody fragment.
  • the present invention provides a recombinant protein that can specifically and rapidly bind to human cardiac muscle-derived troponin I.
  • FIG. 1 shows the antibody.
  • FIG. 2 shows the PCR method in step (c-2).
  • FIG. 1 shows the antibody.
  • antibody 1 has the shape of the “Y” letter.
  • Antibody 1 consists of two Fab regions and one Fc region.
  • Antibody 1 consists of two heavy chains 2 and two light chains 3.
  • Each heavy chain 2 is composed of a heavy chain constant region 1 (reference symbol: 22), a heavy chain constant region 2 (reference symbol: 23), a heavy chain constant region 3 (reference symbol: 24), and a heavy chain variable region 21.
  • Each light chain 3 consists of a light chain variable region 31 and a light chain constant region 32.
  • Each Fab region consists of one heavy chain variable region 21, one heavy chain constant region 1 (reference number: 22), one light chain variable region 31, and one light chain constant region 32.
  • Light chain 3 is linked to heavy chain 2 by linker 4.
  • the heavy chain 2 has a heavy chain variable region 21 at the tip.
  • the light chain 3 has a light chain variable region 31 at the tip.
  • Antigen specifically binds to antibody 1. More specifically, the antigen specifically binds to the Fv region consisting of the heavy chain variable region 21 and the light chain variable region 31.
  • the antigen is troponin I derived from human myocardium.
  • the recombinant protein according to this embodiment includes a light chain variable region 31 consisting of an amino acid sequence represented by SEQ ID NO: 63 and a heavy chain variable region 21 consisting of an amino acid sequence represented by SEQ ID NO: 65. This recombinant protein binds specifically and rapidly to troponin I derived from human myocardium.
  • This recombinant protein is either an antibody or an antibody fragment.
  • Antibody has a “Y” letter shape shown in FIG.
  • the light chain variable region 31 and the heavy chain variable region 21 contained in the antibody have amino acid sequences represented by SEQ ID NO: 63 and SEQ ID NO: 65, respectively.
  • the light chain variable region 31 is linked to the heavy chain variable region 21 via the linker 4.
  • antibody fragments are Fab antibody fragments, F (ab ′) 2 antibody fragments, or scFv antibody fragments.
  • the Fab antibody fragment consists of one Fab region.
  • the Fab antibody fragment consists of one light chain variable region 31 (SEQ ID NO: 63), one heavy chain variable region 21 (SEQ ID NO: 65), one light chain constant region 32, one heavy chain constant region. 1 (reference numeral: 22) and the linker 4.
  • the light chain constant region 32 is linked to the heavy chain constant region 1 (reference number: 22) via the linker 4.
  • An F (ab ′) 2 antibody fragment consists of two Fab regions. As described above, each Fab region has one light chain variable region 31 (SEQ ID NO: 63), one heavy chain variable region 21 (SEQ ID NO: 65), one light chain constant region 32, one heavy chain constant. It consists of a region 1 (reference number: 22) and a linker 4. These two Fab regions are linked to each other via another linker (not shown). Preferably, one heavy chain constant region 1 (reference number: 22) is linked to the other heavy chain constant region 1 (reference number: 22) via another linker (not shown).
  • the scFv antibody fragment consists of a light chain variable region 31 (SEQ ID NO: 63), a heavy chain variable region 21 (SEQ ID NO: 65), and a linker.
  • the light chain variable region 31 (SEQ ID NO: 63) is linked to the heavy chain variable region 21 (SEQ ID NO: 65) via a linker (not shown).
  • the linker is not particularly limited as long as the recombinant protein can specifically and quickly bind to human cardiac muscle-derived troponin I.
  • An example of a linker is a peptide consisting of 5 to 20 amino acids. More specifically, an example of a linker is an amino acid sequence represented by GGGGSGGGGSGGGGS (SEQ ID NO: 64). Another example of a linker is a disulfide bond (-sulfur atom (S) -sulfur atom (S)-).
  • the N-terminus of the light chain variable region 31 (SEQ ID NO: 63) can be modified with an amino acid sequence.
  • the C-terminus can also be modified.
  • the N-terminus of heavy chain variable region 21 can be modified with an amino acid sequence.
  • the C-terminus can also be modified.
  • An example of an amino acid sequence that modifies the C-terminus of the heavy chain variable region 21 (SEQ ID NO: 65) is AAALEHHHHHH (SEQ ID NO: 66).
  • the recombinant protein When the recombinant protein is brought into contact with human cardiac muscle-derived troponin I, the recombinant protein quickly and specifically binds to human cardiac muscle-derived troponin I. More specifically, when the present recombinant protein is mixed with human cardiac muscle-derived troponin I, the recombinant protein quickly and specifically binds to human cardiac muscle-derived troponin I.
  • This recombinant protein can be produced using common protein expression techniques. More specifically, first, a vector containing a gene sequence encoding the present recombinant protein is prepared. An example of a vector is a plasmid. Next, cells (eg, E. coli) are transformed with this vector. The cells are cultured and produce the recombinant protein.
  • a vector containing a gene sequence encoding the present recombinant protein is prepared.
  • An example of a vector is a plasmid.
  • cells eg, E. coli
  • the cells are cultured and produce the recombinant protein.
  • Non-Patent Document 3 discloses a refolding method.
  • Table 1 shows forward mix primers (primers 1 to 21, SEQ ID NOs: 02 to 22) for amplifying the light chain variable region.
  • Table 2 shows forward mix primers (primers 22 to 44, SEQ ID NOs: 23 to 45) for amplifying the heavy chain variable region.
  • Table 3 shows reverse mix primers (primers 45 to 49, SEQ ID NOs: 46 to 50) for amplifying the light chain variable region.
  • Table 4 shows reverse mix primers (primers 50 to 55, SEQ ID NOs: 51 to 56) for amplifying the heavy chain variable region.
  • sulfo-SMCC crosslinker 0.5 mg was dissolved in 100 microliters of phosphate buffered saline to obtain a first aqueous solution. This first aqueous solution was allowed to stand at a temperature of 50 ° C. for 10 minutes.
  • Human serum albumin (10 mg) was dissolved in 1 ml of phosphate buffered saline to obtain a second aqueous solution.
  • the first aqueous solution was mixed with the second aqueous solution to obtain a mixture.
  • the mixture was left for 30 minutes.
  • the sulfo-SMCC crosslinker was conjugated to human serum albumin.
  • the mixture was passed through a column (purchased from GE Healthcare, trade name: PD10) to remove unreacted sulfo-SMCC crosslinker.
  • the amino acid (SEQ ID NO: 01, 1.5 mg) was dissolved in dimethyl sulfoxide (hereinafter referred to as “DMSO”) to obtain a DMSO solution.
  • DMSO dimethyl sulfoxide
  • human serum albumin modified with the amino acid sequence (SEQ ID NO: 01) contained in troponin I was obtained.
  • this human serum albumin is referred to as “troponin-modified HSA”.
  • a mixture of phosphate buffered saline (hereinafter referred to as “PBS”) and troponin-modified HSA was injected into BALB / c mice. This was repeated once more. Thus, over a month, BALB / c mice were immunized with troponin-modified HSA. In other words, by feeding the mixture to BALB / C mice, antibodies against troponin-modified HSA were produced in the body of BALB / c mice.
  • PBS phosphate buffered saline
  • the spleen of the immunized BALB / c mouse was removed. According to the cell fusion method described in Non-Patent Document 4, a hybridoma was obtained. Thereafter, the hybridoma was cultured in a culture solution. The number of hybridomas (cells) after culture was approximately 5 ⁇ 10 6 . The hybridoma thus obtained was able to produce a monoclonal antibody that specifically binds to human cardiac muscle-derived troponin I.
  • Step (a2) Extraction of total mouse RNA from hybridoma cells
  • TRIzol purchased from Invitrogen Corporation
  • the culture solution was subjected to centrifugation at a gravitational acceleration of 117600 m / s 2 at a temperature of 4 ° C. for 15 minutes.
  • the supernatant 500 ⁇ L was obtained.
  • Isopropanol 500 ⁇ L was added to the obtained supernatant and allowed to stand at room temperature for 10 minutes.
  • the culture broth was subjected to centrifugation having the same conditions as described above.
  • a 75% aqueous ethanol solution (1 mL) was added to the resulting precipitate to obtain an ethanol solution.
  • the ethanol solution was subjected to centrifugation at a gravitational acceleration of 73500 m / s 2 for 5 minutes.
  • the precipitate was dried. In this way, total mouse RNA was obtained.
  • Step (b-1) Extraction of mRNA from Total Mouse RNA Using Oligotex TM -dT30 ⁇ Super> mRNA Purification kit (purchased from Takara Bio Inc.) , mRNA was extracted from total mouse RNA obtained in step (a2). Extracted.
  • RNase-free water 100 ⁇ L was injected into one microtube.
  • the microtube was set in a block incubator (purchased from Astech Co., Ltd.) and heated to 70 ° C. for 1 hour.
  • RNA was dissolved in RNase-free water (100 ⁇ L).
  • the mixture was subjected to centrifugation at a gravitational acceleration of 147000 m / s 2 for 5 minutes.
  • the supernatant was removed, and the precipitate was suspended in the washing buffer (350 ⁇ L) included in the kit.
  • the suspension was supplied to the column included in the kit.
  • the column was subjected to centrifugation at a gravitational acceleration of 147000 m / s 2 for 30 seconds.
  • wash buffer 350 ⁇ L was supplied to the column. Again, the column was subjected to centrifugation at a gravitational acceleration of 147000 m / s 2 for 30 seconds.
  • a microtube for sample collection was attached to the bottom of the column.
  • RNase-free water (20 ⁇ L) contained in the microtube was supplied to the column.
  • the column was then subjected to centrifugation at a gravitational acceleration of 147,000 m / s 2 for 3 minutes.
  • RNase-free water (20 ⁇ L) was supplied to the column and the column was subjected to centrifugation at a gravitational acceleration of 147000 m / s 2 for 3 minutes.
  • Step (b-3-1) cDNA contained in the amplification solution of the gene encoding the light chain variable region using cDNA, forward primer 1 to 21 (SEQ ID NO: 02 to 22), and reverse primer 45 to 49 (
  • a gene fragment encoding the light chain variable region of the above monoclonal antibody (SEQ ID NO: 58, hereinafter referred to as “VL gene fragment”) was amplified by PCR using SEQ ID NO: 46-50).
  • the polymerase used in this PCR method was purchased from Takara Bio Inc. under the trade name: TaKaRa Ex Taq Hot start Version.
  • This PCR solution contained the amplified VL gene fragment (SEQ ID NO: 58).
  • Step (b-3-2) cDNA contained in the amplification solution of the gene encoding the heavy chain variable region using cDNA, forward primer 22 to 44 (SEQ ID NO: 23 to 45), and reverse primer 50 to 55 (
  • a gene fragment (SEQ ID NO: 57, hereinafter referred to as “VH gene fragment”) encoding the heavy chain variable region of the monoclonal antibody was amplified by PCR using SEQ ID NO: 51 to 56).
  • the polymerase used in this PCR method was purchased from Takara Bio Inc. under the trade name: TaKaRa Ex Taq Hot start Version.
  • This PCR solution contained the amplified VH gene fragment (SEQ ID NO: 57).
  • the obtained PCR solution was subjected to electrophoresis using a gel containing agarose having a concentration of 2% by weight.
  • Step (b-4) Ligation of VL gene fragment and VH gene fragment
  • the purified VH gene fragment (SEQ ID NO: 57) was ligated to the purified VL gene fragment (SEQ ID NO: 58) by the overlap extension PCR method. It was.
  • a gene fragment (SEQ ID NO: 59, hereinafter referred to as “scFv gene fragment”) encoding the scFv antibody fragment of the monoclonal antibody was obtained.
  • the obtained gene fragment (SEQ ID NO: 59) was modified at its 5 ′ end and 3 ′ end by restriction enzyme sites Nco I and Not I, respectively.
  • Step (c-1) Introduction of gene into vector
  • the scFv gene fragment was ligated to a protein expression vector (purchased from Takara Bio Inc., trade name: pET22b (+)). Details of the ligation are described below.
  • the scFv gene fragment was treated with restriction enzymes Nco I and Not I (both purchased from Takara Bio Inc.).
  • the scFv gene fragment was purified by electrophoresis to obtain an aqueous solution containing the scFv gene fragment.
  • the protein expression vector was also treated with restriction enzymes Nco I and Not I (both purchased from Takara Bio Inc.).
  • the protein expression vector was purified by electrophoresis to obtain an aqueous solution containing the protein expression vector.
  • Escherichia coli purchased from Takara Bio Inc., trade name: DH5 ⁇ competent cell
  • Escherichia coli purchased from Takara Bio Inc., trade name: DH5 ⁇ competent cell
  • the protein expression vector pET22b (+) is removed from the LB liquid medium using a kit (QIAGEN, trade name: QIAprep spin miniprep kit). Extracted.
  • a kit QIAGEN, trade name: QIAprep spin miniprep kit.
  • a signal sequence DNA sequence, protein expression vector pET22b (+)) SEQ ID NO: 60 was removed. In this way, an expression vector encoding a wild type scFv antibody fragment was obtained.
  • 196th guanine (G), 197th adenine (A), 198th thymine (T), 205th adenine (A), 206th thymine (T), 207th adenine ( A), 211th adenine (A), 213th T (thymine), 217th T (thymine), 218th C (cytosine), 219th T (thymine), 226th G (guanine) 227th A (adenine) and 228th T (thymine) are C (cytosine), G (guanine), C (cytosine), C (cytosine), G (guanine), C (cytosine), respectively. , C (cytosine), C (cytosine), C (cytosine), G (guanine), C (cytosine), C (cytosine), G (guanine), C (cytosine).
  • a PCR method using primer 58 (SEQ ID NO: 69), primer 59 (SEQ ID NO: 70), and the expression vector obtained in step (c-1) is used. It was implemented.
  • the primer 58 (SEQ ID NO: 69) was complementary to the gene sequence from the 187th base to the 231st base contained in the scFv gene fragment (SEQ ID NO: 59) except for 14 bases to be replaced.
  • Primer 59 (SEQ ID NO: 70) is a gene sequence from the 187th base to the 231st base contained in the gene fragment complementary to the scFv gene fragment (SEQ ID NO: 59) except for 14 bases to be substituted. Complementary.
  • Step (c-3) Using the vector obtained in the protein acquisition step (c-2) using the vector, E. coli (purchased from Takara Bio Inc., trade name: BL21 (DE3)) was transformed. . The E. coli was then incubated for 16 hours at 37 ° C. on LB plate medium containing ampicillin having a concentration of 100 ⁇ g / mL.
  • the obtained culture broth was subjected to centrifugation at a gravity acceleration of 49000 m / s 2 at a temperature of 4 ° C. for 5 minutes.
  • the precipitate containing E. coli was resuspended in phosphate buffered saline (50 mL).
  • the suspension was subjected to sonication to disrupt E. coli.
  • the solution containing the crushed Escherichia coli was subjected to centrifugation at a gravitational acceleration of 98000 m / s 2 and a temperature of 4 ° C. for 30 minutes. In this way, a precipitate was obtained.
  • the precipitate was washed twice with a phosphate buffered saline containing a surfactant having a concentration of 4% (purchased from Wako Pure Chemical Industries, Ltd., trade name: Triton X-100). In addition, the precipitate was washed with phosphate buffered saline containing no surfactant.
  • the aqueous solution A (10 mL) containing the reagents shown in Table 5 was added to the precipitate.
  • Aqueous solution A had a pH of 6.
  • Aqueous solution A was passed through a filter having a mesh size of 0.45 ⁇ m (purchased from Sartorius, trade name: Minisart) to remove the residue. In this way, a filtrate was obtained.
  • Aqueous solution B had a pH of 8.0. In this way, an aqueous solution having a volume of 3 mL was obtained.
  • the aqueous solution (3 mL) was added dropwise to the aqueous solution B having 1 liter of solution. Thereafter, the obtained aqueous solution was stirred at a temperature of 4 ° C. for 96 hours. In this way, a mutant scFv antibody fragment (SEQ ID NO: 61) was obtained.
  • the solution was concentrated to have a volume of 10 mL using a filtration unit (VIVAFLOW50, purchased from Sartorius).
  • VIPFLOW50 purchased from Sartorius
  • the mutant scFv antibody fragment contained in the solution was purified using a column (purchased from GE Healthcare, trade name: HiLoad 26/60 Superdex 75 pg).
  • Light chain variable region (SEQ ID NO: 63): DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPRRRFRGRGRGTRFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELKRKR Amino acid sequence modified at the N-terminus of the light chain variable region: None Amino acid sequence modified at the C-terminus of the light chain variable region: None Linker (SEQ ID NO: 64): GGGGSGGGGSGGGGS Heavy chain variable region (SEQ ID NO: 65): EVKLEESGGGLVKPGGTLKLSCAASGFFTFSYAMSWVRQTPEKRLLDWATISSGGGSYIFYPDSVKGRFTISRQRNSLLYLQMNSLRSTEDTAMYYCARHNPDKSGFAYWGQGTLVTVSA
  • Step (d) Calculation of binding rate constant and dissociation rate constant Using the molecular interaction analyzer Biacore T100 (purchased from GE Healthcare), the affinity of the mutant scFv antibody fragment is determined by the molecular interaction analyzer Biacore. It was calculated according to the manual attached to T100.
  • CM5 chip Human myocardial troponin I (purchased from Funakoshi) having approximately 500 RUs (Resonance Units) was immobilized on a CM5 chip (purchased from GE Healthcare). This CM5 chip was set in Biacore T100. Next, an aqueous solution containing the mutant scFv antibody fragment (concentrations: 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM, volume: 150 microliters) was flowed into Biacore T100. Table 8 shows dissociation constants measured by the intermolecular interaction analyzer Biacore T100.
  • Comparative Example 1 In Comparative Example 1, an experiment similar to Example 1 was performed, except that the step (c-2) was not performed. In this way, a wild type scFv antibody fragment consisting of the amino acid sequence represented by SEQ ID NO: 62 was obtained. Similar to Example 1, the dissociation constant (KD) of the wild type scFv antibody fragment was measured. The results are shown in Table 8.
  • the mutant scFv antibody fragment according to Example 1 has a higher affinity than the wild scFv antibody fragment according to Comparative Example 1. This means that the mutant scFv antibody fragment specifically binds to human cardiac muscle-derived troponin I more rapidly than the wild scFv antibody fragment.
  • the method according to the present invention can be used in a sensor for detecting acute myocardial infarction.
  • Antibody 2 Heavy chain 21: Heavy chain variable region 22: Heavy chain constant region 1 23: Heavy chain constant region 2 24: Heavy chain constant region 3 3: Light chain 31: Light chain variable region 32: Light chain constant region 32 4: Linker

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Abstract

La présente invention concerne une protéine recombinée capable de se lier de façon spécifique et rapide à la troponine I du tissu myocardique humain. La présente invention concerne, en particulier, une protéine recombinée capable de se lier de façon spécifique et rapide à la troponine I du tissu myocardique humain, ladite protéine recombinée comportant une région variable de chaîne légère constituée d'une séquence d'acides aminés représentée par SEQ ID NO. 63 et une région variable de chaîne lourde constituée d'une séquence d'acides aminés représentée par SEQ ID NO. 65. La présente invention concerne également un procédé de liaison de ladite protéine recombinée à la troponine I du tissu myocardique humain, ledit procédé comprenant une étape (a) consistant à préparer ladite protéine recombinée et une étape (b) consistant à amener la troponine I du tissu myocardique humain en contact avec la protéine recombinée et à lier ladite protéine recombinée à la troponine I du tissu myocardique humain.
PCT/JP2014/001219 2013-05-29 2014-03-05 Protéine recombinée capable de se lier de façon spécifique et rapide à la troponine i du tissu myocardique humain WO2014192202A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012518418A (ja) * 2009-02-24 2012-08-16 アボット・ラボラトリーズ トロポニンｉに対する抗体およびこれらの使用法
JP2013029441A (ja) * 2011-07-29 2013-02-07 Panasonic Corp トロポニンｉの測定方法
WO2013094080A1 (fr) * 2011-12-20 2013-06-27 パナソニック株式会社 Protéine recombinante apte à se lier spécifiquement et rapidement à la troponine-i issue des muscles cardiaques humains
WO2013099043A1 (fr) * 2011-12-26 2013-07-04 パナソニック株式会社 Protéine de recombinaison capable de se lier spécifiquement et rapidement à une troponine-i des muscles cardiaques humains
WO2013175678A1 (fr) * 2012-05-24 2013-11-28 パナソニック株式会社 Protéine variante pouvant se lier spécifiquement et rapidement à la troponine cardiaque humaine i

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012518418A (ja) * 2009-02-24 2012-08-16 アボット・ラボラトリーズ トロポニンｉに対する抗体およびこれらの使用法
JP2013029441A (ja) * 2011-07-29 2013-02-07 Panasonic Corp トロポニンｉの測定方法
WO2013094080A1 (fr) * 2011-12-20 2013-06-27 パナソニック株式会社 Protéine recombinante apte à se lier spécifiquement et rapidement à la troponine-i issue des muscles cardiaques humains
WO2013099043A1 (fr) * 2011-12-26 2013-07-04 パナソニック株式会社 Protéine de recombinaison capable de se lier spécifiquement et rapidement à une troponine-i des muscles cardiaques humains
WO2013175678A1 (fr) * 2012-05-24 2013-11-28 パナソニック株式会社 Protéine variante pouvant se lier spécifiquement et rapidement à la troponine cardiaque humaine i

Non-Patent Citations (2)

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Title
ALTSHULER E.: "Comparison of biochemical and immunochemical properties of the recombinant scFv and Fab fragments of antibodies TnIMAB specific to human cardiac troponin I", FEBS JOURNAL, vol. 279, no. SUPPL., 2012, pages 335, P 14 - 20 *
FUKUNAGA A. ET AL.: "Improving the affinity of an antibody for its antigen via long-range electrostatic interactions", PROTEIN ENGINEERING, vol. 26, no. 12, pages 773 - 780 *

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