JP6174778B1 - Liver-type fatty acid binding protein preparation, method for evaluating the preparation, method for suppressing the fluctuation range of the measurement value caused by liver-type fatty acid binding protein in the measurement using the preparation, calibration curve for liver-type fatty acid binding protein Method for preparing and method for quantifying the protein - Google Patents

Abstract

[Problem] To provide a liver type fatty acid binding protein preparation capable of reducing the fluctuation range of the measurement value caused by the liver type fatty acid binding protein in the measurement using a substance that specifically binds. A liver comprising an amino acid sequence having 90% or more identity with a specific sequence of a fatty acid binding protein, wherein the oxidation rate of the 19th methionine is 30% or more, or the 113th methionine has an oxidation rate of 70% or more. Liver type fatty acid binding protein preparation containing type fatty acid binding protein. At least one fatty acid selected from the group consisting of arachidonic acid, oleic acid, 8-isoprostaglandin F2α and 2,3-dinor-8-isoprostaglandin F2α with respect to the molar amount of liver-type fatty acid binding protein. A liver type fatty acid binding protein preparation comprising a liver type fatty acid binding protein comprising a 30% or more molar amount and an amino acid sequence having 90% or more identity with the specific sequence. [Selection] Figure 1

Description

  The present invention provides a liver-type fatty acid binding protein preparation that can reduce the fluctuation range of the measurement value caused by the liver-type fatty acid binding protein in the measurement using a substance that specifically binds, a method for evaluating the preparation, The present invention relates to a method for suppressing a fluctuation range of a measurement value caused by liver-type fatty acid binding protein in measurement using a product, a method for preparing a calibration curve of liver-type fatty acid binding protein, and a method for quantifying the protein.

Fatty Acid-Binding Protein (hereinafter also referred to as FABP) is a protein having a molecular weight of about 14 kDa belonging to the family of intracellular lipid-binding proteins, and reversibly binds to hydrophobic ligands including fatty acids to transport into the cell. It is known to bear (for example, Non-Patent Document 1). Among them, hepatic fatty acid binding protein (L-type fatty acid-binding protein; hereinafter also simply referred to as “L-FABP protein”) is localized in the cytoplasm of the proximal tubule cells of the liver and kidney, and urine The amount of urinary excretion increases in response to ischemia / oxidative stress due to tubule injury (for example, Non-Patent Document 2). Therefore, it is possible to test for renal diseases by detecting L-FABP protein derived from kidney tissue in urine (for example, Patent Document 1).
In addition, as shown in FIG. 18, the L-FABP protein is stabilized in such a way that two α-helices cover a β-barrel structure in which two antiparallel β-sheets are perpendicular to each other. It is known to bind to free fatty acids (for example, oleic acid) (PDB ID: 2LKK) (Non-patent Document 3).
The L-FABP protein has a mechanism for transporting free fatty acids to mitochondria and peroxisomes and promoting β-oxidation (Non-patent Document 4).
The binding affinity of the fatty acid ligand tends to increase as the carbon chain of the fatty acid grows and double bonds increase (Non-Patent Documents 5 and 6). It has been reported that the binding affinity is high (Non-patent Document 7).
In addition, a report on the study of the antioxidant mechanism of L-FABP protein indicates that the methionine residue of rat L-FABP protein was oxidized by AAPH (Non-patent Document 8).

JP-A-11-242026

Furuhashi, M .; , Et al. : Nat Rev Drug Discov, 7: 489-503, 2008 Kamijo, A .; et al. : J Lab Clin Med, 143: 23-30, 2004 Cai, J .; et al. : Biophys J, 102: 2585-2594, 2012 Veerkamp, J. et al. H. et al. : Prostaglandins Leukot Essent Fatty Acids, 49: 887-906, 1993. Zimmerman, A.M. W. et al. : Int J Biochem Cell Biol, 33: 865-876, 2001 Norris, A.M. W. , Spector, A .; A. : J Lipid Res, 43: 646-653, 2002 Raza, H .; et al. : Biochem Biophys Res Commun, 161: 448-455, 1989. Yan, J .; et al. : J Lipid Res, 50: 2445-2454, 2009

As a test kit for detecting the L-FABP protein as described above, for example, a test kit employing a sandwich ELISA method in which two types of antibodies having different recognition sites for the L-FABP protein are used has been developed. .
Here, FIG. 1 is a diagram showing the storage stability of L-FABP, and the graph in FIG. 1 shows changes in ELISA measurement values depending on the storage days when L-FABP is stored at 4 ° C., 25 ° C. or 37 ° C. (Ratio (%) when the sample stored at −80 ° C. is 100).
In the above-described test kit employing the sandwich ELISA method, with the conventional measurement standard substance (standard) using recombinant L-FABP protein, when the L-FABP protein is stored for a long time at room temperature (25 ° C.) or more, FIG. As shown, the antibody binding ability was changed, and there was a problem that L-FABP protein could not be accurately measured by an immunological technique using an antigen-antibody reaction such as ELISA.
Due to the instability of the L-FABP protein, strict control is required for the production conditions and measurement environment of the L-FABP protein preparation, and the preparation is required to be stored at a low temperature for further operability and stability. Improvement was desired.

  The present invention has been made in view of the above circumstances, and a liver-type fatty acid binding protein preparation capable of reducing the fluctuation range of the measurement value caused by the liver-type fatty acid binding protein in the measurement using a substance that specifically binds. , A method for evaluating the preparation, a method for suppressing the fluctuation range of the measurement value caused by the liver-type fatty acid binding protein in the measurement using the preparation, a method for preparing a calibration curve for the liver-type fatty acid binding protein, and the protein The purpose is to provide a method for quantifying

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the L-FABP protein is treated with 2,2′-azobis-2-amidinopropane (hereinafter abbreviated as AAPH) which is an oxidant. As a result, it was found that the antibody binding ability in ELISA was changed, and the ELISA measurement value varied greatly. Surprisingly, even when AAPH was not added (added with H ₂ O), the ELISA measurement value was increased by reacting at room temperature for 1 hour, and it was also found that the effect of air oxidation occurred. . In particular, regarding the suppression of the fluctuation range of the ELISA measurement value, it has been found that the oxidation rate of the 19th and 113th methionine is dominant among the 19th, 74th and 113th methionine.

Furthermore, the L-FABP protein, in which the methionine residue in the L-FABP protein is mutated to another amino acid by genetic modification technology, changes its antibody binding ability even during oxidation reaction, addition of fatty acids, and long-term storage at temperatures above room temperature. It was found that it can be stabilized without.
The present invention has been completed based on the above findings.

That is, the present invention is as follows.
The first aspect of the present invention is:
A liver type fatty acid binding protein comprising an amino acid sequence having 90% or more identity with SEQ ID NO: 1 in the sequence listing, wherein the oxidation rate of the 19th methionine is 30% or more, or the 113th methionine has an oxidation rate of 70% or more. Contains liver-type fatty acid binding protein preparation.
The second aspect of the present invention is:
Relative molar amount of liver-type fatty acid binding protein, arachidonic acid, oleic acid, 8 iso static Prostaglandin F _2.alpha and 2,3-dinor-8-iso static prostaglandin F least one selected from the group consisting of _2.alpha Is a liver-type fatty acid-binding protein preparation comprising a liver-type fatty acid-binding protein comprising a 30-fold or higher molar amount of the fatty acid and having an amino acid sequence having 90% or more identity with SEQ ID NO: 1 in the Sequence Listing.

The third aspect of the present invention is:
It is a liver type fatty acid binding protein preparation with an oxidation variation coefficient set to 1.4 or less.
The fourth aspect of the present invention is:
This is a method for evaluating a liver-type fatty acid binding protein preparation using an oxidation coefficient of variation as an index.
According to a fifth aspect of the present invention,
A method for suppressing a fluctuation range of a measurement value in a measurement using a liver type fatty acid binding protein preparation,
The oxidation rate of the 19th methionine of liver type fatty acid binding protein is 30% or more, or the oxidation rate of the 113th methionine is 70% or more, and arachidonic acid, oleic acid, 8-isoprostaglandin F _2α and At least one selected from the group consisting of containing at least one fatty acid selected from the group consisting of 2,3-dinor-8-isoprostaglandin F _{2α in the} liver type fatty acid binding protein preparation It is the method of including.
The sixth aspect of the present invention is:
A method for preparing a calibration curve for a liver-type fatty acid binding protein using the liver-type fatty acid binding protein preparation according to the first or second aspect.
The seventh aspect of the present invention is
It is a method for quantifying liver-type fatty acid binding protein in a sample using a calibration curve prepared by the method according to the sixth aspect.

According to the present invention, a liver-type fatty acid-binding protein preparation, a calibration curve for liver-type fatty acid-binding protein, which can reduce the fluctuation range of the measurement value caused by the liver-type fatty acid binding protein in the measurement using a substance that specifically binds Can be provided, and a method for quantifying the protein can be provided.
Since the liver-type fatty acid binding protein preparation of the present invention can suppress changes in antibody binding ability due to oxidation or fatty acid binding state, it does not depend on the source species or protein expression method. That is, the liver type fatty acid binding protein preparation of the present invention can provide stability and versatility in production conditions, storage conditions, and operability.
Moreover, the method for evaluating the liver-type fatty acid binding protein preparation of the present invention can evaluate the degree to which the measured value of the liver-type fatty acid binding protein varies due to oxidation as a coefficient.

Since the liver type fatty acid binding protein preparation of the present invention can reduce the fluctuation range of the measurement value caused by the liver type fatty acid binding protein in the measurement using a substance that specifically binds (for example, an antibody), Enables accurate detection or quantification regardless of the degree of oxidation.
In addition, since the liver type fatty acid binding protein preparation of the present invention can reduce the fluctuation range of the measurement value caused by the liver type fatty acid binding protein in the measurement using a substance that specifically binds, It is expected to facilitate the production management of liver-type fatty acid binding protein as a quality control substance, and to improve the operability and versatility of the measurement in the immunological technique.

It is a figure which shows the storage stability of L-FABP protein. (A) is a figure which shows the amino acid sequence of human L-FABP protein shown to sequence number 1, (b) is a figure which shows the estimated molecular weight of the various peptide fragments which can be produced by the trypsin digestion of human L-FABP protein. It is. (A) shows peptide fragment No. 1 containing Met1 obtained by trypsin digesting the human L-FABP protein after reaction with AAPH at various concentrations (40 mM, 200 mM). 1 (b) is a peptide fragment No. 1 containing Met19 obtained by trypsin digestion of human L-FABP protein after reaction with AAPH at various concentrations. (C) is a peptide fragment No. 2 containing Met74 obtained by trypsin digestion of the human L-FABP protein after reaction with AAPH at various concentrations. 9 and peptide fragment no. 10 (d) is a diagram showing the MS spectrum of No. 10, wherein (d) shows peptide fragment No. 1 containing Met113 obtained by trypsin digestion of human L-FABP protein after reaction with AAPH at various concentrations. 14 shows MS spectra of 14 and peptide fragment 15. FIG. It is a figure which shows the change (ratio (%) when the case of non-processing is set to 100) of the ELISA measurement value of the variation | mutation L-FABP protein by AAPH treatment of various density | concentrations. It is a figure which shows the change (ratio (%) which made the unprocessed sample 100) by the ELISA measurement value by the AAPH process of the mutant L-FABP protein. It is a figure which shows the influence (ratio (%) which set the untreated sample to 100) in the ELISA measurement using the labeled antibody from which the recognition site | part differs from the clone 2. FIG. It is the figure which measured the oxidation rate of each 19th, 74th, and 113th methionine of various L-FABP protein from which progress of oxidation differs. It is a figure which shows the change (ratio (%) which set the 4 degreeC preservation | save sample to 100) by the 37 degreeC two-week preservation | save of the oxidized L-FABP protein. It is a figure which shows the change (ratio (%) which made the amount of fatty acid addition 0 mol times 100 be 100) of the ELISA measured value by various fatty acids of various density | concentrations. It is a figure which shows the change (ratio (%) which set the unprocessed sample to 100) by the ELISA measurement value by the fatty acid addition process of L-FABP mutein. In the absence of BSA, after addition of arachidonic acid, the change in arachidonic acid addition amount and ELISA measured value when free arachidonic acid was removed by dialysis (rate (%) where arachidonic acid addition amount was 0 mol times 100) FIG. In the absence of BSA, oleic acid was added and free oleic acid was removed by dialysis. Changes in oleic acid addition amount and ELISA measurement value (ratio (%) where the oleic acid addition amount was 0 mol times 100) FIG. In the absence of BSA, a 50-fold molar amount of arachidonic acid was added, free arachidonic acid was removed by dialysis, and changes in ELISA measured values by storage of L-FABP protein bound with arachidonic acid at 37 ° C. for 2 weeks (4 ° C. It is a figure which shows the ratio (%) which made the preservation | save sample 100. In the absence of BSA, 1000 mol-fold amount of oleic acid was added, free oleic acid was removed by dialysis, and changes in ELISA measurement values due to storage of oleic acid-bound L-FABP protein at 37 ° C for 2 weeks (4 ° C It is a figure which shows the ratio (%) which made the preservation | save sample 100. It is a figure which shows an oxidation variation coefficient about L-FABP WT processed with L-FABP M19L / M74L / M113L, 0.5 mM or 4 mM AAPH, and L-FABP WT. It is a figure which shows the correlation with the content rate of residual unoxidized methionine, and an oxidation fluctuation coefficient about each of the 19th, 74th, and 113th methionine. It is a figure which shows the calculation result of the oxidation fluctuation coefficient of various L-FABP. It is a figure which shows the three-dimensional structure model which shows the coupling | bonding with the free fatty acid of L-FABP protein.

  Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be implemented with appropriate modifications within the scope of the object of the present invention. .

(Mutant liver type fatty acid binding protein)
Mutant liver type fatty acid binding protein is
It consists of an amino acid sequence having 90% or more identity with SEQ ID NO: 1 in the sequence listing, and it is preferable that one or more methionines at the 19th, 74th, and 113th positions are substituted with nonpolar amino acids other than methionine.
The mutant liver type fatty acid binding protein is a substance that specifically binds to a liver type fatty acid binding protein (for example, by replacing one or more methionines at the 19th, 74th, and 113th positions with a non-polar amino acid other than methionine) The binding ability of the antibody) can be stabilized.
In addition to methionine, oxidative modifications such as cysteine residues that undergo direct addition of disulfide bonds and oxygen, lysine residues that are carbonylated, arginine residues, proline residues, and tyrosine residues that are nitrated are known. (Toda T., et al., Basic Aging Research, 35 (3); 17-22, 2011), the amino acid replacing one or more methionines at the 19th, 74th, and 113th is a nonpolar amino acid. Can prevent oxidation modification.

SEQ ID NO: 1 represents the amino acid sequence of wild-type human L-FABP protein (hereinafter also referred to as L-FABP WT).
As used herein, “amino acid sequence having 90% or more identity” means that amino acid identity is 90% or more, and identity is preferably 95% or more, more preferably 97% or more. .
Even if it is a mutated protein due to substitution, insertion, deletion, etc. on the amino acid sequence of the wild-type human liver type fatty acid binding protein shown in SEQ ID NO: 1 in the sequence listing, the mutation is 3 of the wild type human liver type fatty acid binding protein. All of these mutations that are highly conserved in the dimensional structure may belong to the liver type fatty acid binding protein.
The side chains of amino acids that constitute protein components differ in hydrophobicity, charge, size, etc., but mean that they do not substantially affect the three-dimensional structure (also referred to as a three-dimensional structure) of the entire protein. Some of the relationships that are highly conserved are known empirically and by physicochemical measurements. For example, for substitution of amino acid residues, glycine (Gly) and proline (Pro), Gly and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln) ), Aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), Thr and serine (Ser) or Ala, lysine (Lys) and arginine (Arg), and the like.

The method for obtaining the mutant liver fatty acid binding protein is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by a gene recombination technique.
Since the amino acid sequence and gene sequence of L-FABP protein have already been reported (Veerkamp and Maatman, Prog. Lipid Res., 34: 17-52, 1995), for example, primers are designed based on them, and PCR method The above-mentioned mutant liver-type fatty acid-binding protein can be prepared by cloning cDNA from an appropriate cDNA library or the like and carrying out gene recombination using the cDNA.

  In the mutant liver type fatty acid binding protein, it is preferable that two or more methionines at the 19th, 74th and 113th are substituted with nonpolar amino acids other than methionine, and two or more methionines containing the 19th methionine are methionine. It is more preferable that the amino acid is substituted with a nonpolar amino acid other than methionine, and it is further preferable that all of the 19th, 74th and 113th methionines are substituted with nonpolar amino acids other than methionine.

  As the nonpolar amino acid replacing one or more methionines at the 19th, 74th, and 113th, one or more methionines at the 19th, 74th, and 113th may be substituted with the same nonpolar amino acid, It may be substituted with a different nonpolar amino acid. The nonpolar amino acid that replaces one or more methionines at the 19th, 74th, and 113th positions is preferably leucine, isoleucine, valine, alanine, phenylalanine, tryptophan, and methionine that does not significantly change antibody binding From the viewpoint of a similar structure, leucine, isoleucine, valine and alanine are more preferable, leucine, isoleucine and valine are more preferable, leucine and isoleucine are particularly preferable, and leucine is most preferable. preferable.

(DNA encoding mutant liver fatty acid binding protein)
The DNA (mutant gene) encoding the mutant liver fatty acid-binding protein can be prepared by any method such as chemical synthesis, genetic engineering, or mutagenesis. As described above, since the amino acid sequence and gene sequence of the L-FABP protein have already been reported, for example, primers are designed based on them, and cDNA is cloned from an appropriate cDNA library or the like by the PCR method. And can be obtained by genetic recombination. Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular cloning 2nd edition, Current Protocols in Molecular. It can be performed according to the method described in biology and the like.

(Transformed cells)
A transformed cell can be prepared by introducing the DNA or a recombinant vector containing the DNA into an appropriate host.
The recombinant vector containing the DNA can be prepared by a general genetic recombination technique using an appropriate host vector system. As suitable vectors, plasmids derived from E. coli (eg, pBR322, pUC118, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pSH19, etc.), animal viruses such as bacteriophages, retroviruses, vaccinia viruses and the like can be used. .
Examples of the host cell into which the DNA or the recombinant vector containing the DNA is introduced include bacteria and yeast.
Examples of bacterial cells include Gram-positive bacteria such as Corynebacterium bacteria (eg Corynebacterium glutamicum), Bacillus bacteria (eg Bacillus subtilis) or Streptomyces bacteria, or Gram-negative bacteria such as Escherichia coli. Is mentioned. Transformation of these bacteria may be performed by using competent cells by a protoplast method or a known method.
Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, such as Saccharomyces cerevisiae or Saccharomyces kluyveri. Examples of the method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroblast method, and a lithium acetate method.
As the cells transformed with the DNA, Corynebacterium glutamicum is used because the mutant liver fatty acid-binding protein is efficiently produced and purification of the liver-type fatty acid binding protein described below does not require complicated steps. It is preferably a protein secretion expression system (CORYNEX (registered trademark): manufactured by Ajinomoto Co., Inc.).

(Method for producing the above-mentioned mutant liver type fatty acid binding protein)
The method for producing a mutant liver fatty acid-binding protein preferably includes a step of culturing the transformed cell and recovering the protein.
The transformed cells are cultured in a suitable nutrient medium under conditions that allow expression of the introduced gene. In order to recover the protein from the culture of the transformed cells, ordinary protein isolation and purification methods may be used.
For example, when the above protein is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer solution, disrupted by an ultrasonic disrupter, etc. Obtain an extract. From the supernatant obtained by centrifuging the cell-free extract, a normal protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) Sepharose, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), resin such as butyl sepharose and phenyl sepharose Use a hydrophobic chromatography method, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing etc. alone or in combination to obtain a purified preparation be able to.
By using the protein secretion expression system using the above-mentioned Corynebacterium glutamicum (CORYNEX (registered trademark): manufactured by Ajinomoto Co., Inc.), there is no need for complicated steps such as crushing cells to obtain a cell-free extract. After the centrifugation, a purified sample can be obtained by purification by anion exchange chromatography (for example, HiTrapQ FF 5 mL FPLC column).

<Liver type fatty acid binding protein preparation>
In this specification, “standard” refers to measurement standard substances (calibrators) and quality control substances (controls) as representative examples, and commonly used reference standard substances, practical standard substances, manufacturer product calibration substances, and diagnostic standards. It means all substances including standard substances such as substances and calibration standards, and other substances including quality control substances (Yoshiaki Iizuka et al., “Current Status of Traceability Chain and Uncertainty” Biological Sample Analysis Vol. 34 , No3 (2011), 179-188, Japanese Pharmacopoeia Standards and Reference Substances, 2009 JCCLS (Japan Clinical Laboratory Standards Association) Terminology Committee Terminology (English) and its Japanese translation (draft) 226).
The liver-type fatty acid binding protein preparation according to the first aspect of the present invention comprises an amino acid sequence having 90% or more identity with SEQ ID NO: 1 in the Sequence Listing, and the oxidation rate of the 19th methionine is 30% or more, or 113. The second methionine contains a liver type fatty acid binding protein having an oxidation rate of 70% or more.
As will be described later with reference to FIG. 7, when the oxidation rate of the 19th methionine is 30% or more, or the 113th methionine has an oxidation rate of 70% or more, the increase in the oxidation rate is further suppressed. , Fluctuations in binding ability due to a substance that specifically binds can be suppressed.
In the liver-type fatty acid binding protein preparation according to the first aspect, when the 19th methionine oxidation rate is 30% or more, from the viewpoint that fluctuations in binding ability due to a specifically binding substance can be further suppressed, At least one methionine selected from the group consisting of 74th and 113th may or may not be substituted with the above-mentioned nonpolar amino acid other than methionine.
From the same viewpoint, when the oxidation rate of the 113th methionine is 70% or more, at least one methionine selected from the group consisting of the 19th and 74th is substituted with the above-mentioned nonpolar amino acid other than methionine. It does not have to be.
In addition, the oxidation rate of the 19th methionine is preferably 35% or more, more preferably 38% or more, from the viewpoint that fluctuations in binding ability due to a substance that specifically binds can be further suppressed. % Or more is more preferable, and 45% or more is particularly preferable.
From the same viewpoint, the oxidation rate of the 113th methionine is preferably 73% or more, more preferably 75% or more, and further preferably 80% or more.
As the method for measuring the oxidation rate, for example, an MS spectrum of a peptide fragment containing Met19 in FIG. 3B described later, an MS spectrum of a peptide fragment containing Met74 in FIG. 3C, and Met113 in FIG. It can be calculated from a comparison between the peak in the MS spectrum of the peptide fragment containing no oxidation and the peak after the oxidation treatment.
In addition, the liver type fatty acid binding protein preparation is not dependent on the oxidation rate of the 19th methionine and the oxidation rate of the 113th methionine, from the viewpoint that fluctuations in binding ability due to a specifically binding substance can be suppressed. The oxidation rate of the 74th methionine may be 60% or more.
In this case, the oxidation rate of the 74th methionine is preferably 65% or more, more preferably the oxidation rate of the 74th methionine is 70% or more, and the oxidation rate of the 74th methionine is 75% or more. More preferably, the oxidation rate of the 74th methionine is particularly preferably 80% or more, and the oxidation rate of the 74th methionine is most preferably 85% or more.
The liver-type fatty acid binding protein preparation having the oxidation rate can be produced using an oxidizing agent such as AAPH or can be produced by air oxidation.

In addition, the liver type fatty acid binding protein preparation according to the second aspect is arachidonic acid, oleic acid, 8-isoprostaglandin F _2α and 2,3-dinor-8 with respect to the molar amount of the liver type fatty acid binding protein. -Liver-type fatty acid binding comprising 30-fold or more molar amount of at least one fatty acid selected from the group consisting of isoprostaglandin F _2α and having an amino acid sequence having 90% or more identity with SEQ ID NO: 1 in the sequence listing A liver-type fatty acid binding protein preparation containing protein.
As will be described later with reference to FIG. 9, the present inventors have found that the antibody-binding ability of the L-FABP protein varies depending on the type (FIG. 9 (b)) and concentration of the fatty acid that binds to the L-FABP protein. I found it.
The liver-type fatty acid-binding protein preparation according to the second aspect includes arachidonic acid, oleic acid, 8-isoprostaglandin F _2α, and 2,3-dinor-8-isoproprote with respect to the molar amount of the liver-type fatty acid binding protein. When at least one fatty acid selected from the group consisting of staglandin F _2α is included, when a plurality of fatty acids are contained, the total content thereof is 30 times or more in molar amount, thereby expressing L-FABP protein as a standard product Or in the assay system used as a sample, the type of fatty acid (for example, arachidonic acid, oleic acid, 8-isoprostaglandin) bound by the expression system or expression site or organ from different species of origin. F _2.alpha and 2,3-dinor-8-iso static prostaglandin _{F 2.alpha} etc.), binding amount, even if the degree of peroxidation are different, specifically binds Variations in binding ability due to the quality been suppressed, reference material for measuring, can function as a standard, such as control material.
From the viewpoint of further suppressing the change in binding ability due to the substance that specifically binds, arachidonic acid, oleic acid, 8-isoprostaglandin F _2α and 2,3-dinor are used relative to the molar amount of the liver-type fatty acid binding protein. It is preferable that at least one fatty acid selected from the group consisting of -8-isoprostaglandin F _2α is contained in a molar amount of 50 times or more as a total content when the number of fatty acids is plural, and is 75 times or more. More preferably, it is more preferably 100 times or more, and particularly preferably 300 times or more.

More specifically, when the fatty acid is arachidonic acid, the molar amount of the liver-type fatty acid binding protein is preferably 30 times or more, more preferably 50 times or more, and more preferably 75 times or more. It is more preferable to include a molar amount of, and particularly preferable to include a molar amount of 100 times or more.
When the fatty acid is oleic acid, the molar amount of the liver-type fatty acid binding protein is preferably 100 times or more, more preferably 300 times or more, and more preferably 1000 times or more. It is more preferable to include.
In addition, when the fatty acid is 8-isoprostaglandin F _2α , the molar amount of the liver-type fatty acid binding protein is preferably 500 times or more, more preferably 1000 times or more.

In addition, when the liver type fatty acid binding protein preparation according to the second embodiment contains bovine serum albumin (BSA) as an adsorption inhibitor, arachidone is used with respect to the molar amount of BSA from the viewpoint of adsorption of fatty acid on BSA. At least one fatty acid selected from the group consisting of acid, oleic acid, 8-isoprostaglandin F _2α and 2,3-dinor-8-isoprostaglandin F _2α , in the case where there are a plurality of fatty acids The content is preferably 0.02 to 10 times the molar amount, more preferably 0.2 to 5 times the molar amount.
In addition, when the preparation contains BSA and the fatty acid is arachidonic acid, arachidonic acid is preferably included in a molar amount of 1000 times or more, and preferably in a molar amount of 10,000 times or more with respect to the molar amount of liver type fatty acid binding protein. More preferably, it is more preferable to include a molar amount of 100,000 times or more, and particularly preferable to include a molar amount of 200,000 times or more.
When the preparation contains BSA and the fatty acid is oleic acid, oleic acid is preferably contained in a molar amount of 100,000 times or more, and a molar amount of 200,000 times or more, relative to the molar amount of liver-type fatty acid binding protein. More preferred.
When the preparation contains BSA and the fatty acid is 8-isoprostaglandin F _2α , 8-isoprostaglandin F _2α should contain a molar amount of 200,000 times or more with respect to the molar amount of liver-type fatty acid binding protein. Is preferred.
The liver-type fatty acid binding protein preparation according to the second aspect containing a specific amount of the at least one fatty acid is a liquid containing the liver-type fatty acid binding protein preparation in a molar amount of the liver type fatty acid binding protein, The above-mentioned at least one fatty acid is prepared by adding a molar amount of 30 times or more as a total content when there are a plurality of types of fatty acids, or by cell culture, protein isolation or purification conditions to achieve such a content. be able to. The content of the at least one fatty acid corresponds to the amount added.
The upper limit of the fatty acid content is not particularly limited, but when the following adsorption inhibitor is further contained, the fatty acid may be adsorbed to the adsorption inhibitor, so that the molar amount of liver fatty acid binding protein The above fatty acid can be contained in a molar amount of 500,000 times, preferably 300,000 times or less, more preferably 200000 times or less, more preferably 10,000 times or less. Is more preferable.

The liver-type fatty acid binding protein preparation according to the first or second aspect described above has a fluctuation range around 37 ° C. for 2 weeks in a measurement value using a substance that specifically binds to the liver-type fatty acid binding protein (for example, The fluctuation range due to oxidation is not particularly limited as long as the effect of the present invention is not impaired, but is preferably 15% or less, more preferably 10% or less, and still more preferably 5% or less. 1% or less is particularly preferable.
The liver-type fatty acid binding protein preparation according to the first or second aspect may contain an adsorption inhibitor, an arbitrary buffer, an optional surfactant, and the like as necessary.
The adsorption inhibitor is not particularly limited as long as the effects of the present invention are not impaired, and examples thereof include BSA, casein, skim milk, polyethylene glycol and the like, and BSA is preferable.
The content of the adsorption inhibitor in the liver-type fatty acid binding protein preparation according to the first or second aspect is not particularly limited as long as the effect of the present invention is not impaired, but is 0.05 to 10% by mass. It is preferable.

The liver-type fatty acid binding protein preparation according to the first or second aspect is preferably a liver-type fatty acid binding protein preparation for sale from the viewpoint of practical and commercial use. A liver-type fatty acid binding protein preparation to be sold is not a protein that has already been sold, specifically a liver-type fatty acid binding protein preparation that has been left for a long time after the sale.
The liver-type fatty acid binding protein preparation according to the first or second aspect is a measurement standard substance for a kit for measuring liver-type fatty acid binding protein in a sample by an immunological technique using an antigen-antibody reaction, or Standard substance for measuring or detecting L-FABP protein, which is useful as a quality control substance and uses specific binding by anti-L-FABP protein antibody that specifically binds to L-FABP protein (standard) ) Is preferably used.
Examples of the measurement such as detection or quantification of L-FABP protein in which the liver-type fatty acid binding protein preparation according to the first or second aspect is used include enzyme immunoassay (EIA, ELISA), fluorescent enzyme immunoassay ( FLEIA), chemiluminescence enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), immunochromatography (ICA), latex agglutination (LA), fluorescent antibody method (FA), radioimmunoassay (RIA), Western blot (WB), immunoblot, and other assays are used. Two types of antibodies with different recognition sites for antigen (L-FABP protein) are used in combination. It is preferable that the assay employs a sandwich ELISA method.
It is preferable to use one of the two types of antibodies having different recognition sites as a solid-phased antibody bound to the surface in the well of the microplate and the other as a labeled antibody for detection or quantification. There is no restriction | limiting in particular as a label in the said labeled antibody, For example, enzyme labels, such as a peroxidase label, a fluorescent label, an ultraviolet-ray label, a radiation label etc. are mentioned.

  Examples of antibodies having different recognition sites for antigen (L-FABP protein) include antibodies comprising an antibody selected from the group consisting of anti-L-FABP antibody clone 1, clone 2, clone L and clone F, and anti-L- A combination including FABP antibody clone L or a combination including anti-L-FABP antibody clone 2 is preferable, a combination including anti-L-FABP antibody clone L is more preferable, and anti-L-FABP antibody clone L is It is more preferable to use any anti-L-FABP antibody as a labeled antibody, using as a solid-phased antibody, using anti-L-FABP antibody clone L as a solid-phased antibody, and using anti-L-FABP antibody clone 2 as a labeled antibody. It is particularly preferable to use it.

The L-FABP protein measurement kit used in such an assay includes the liver type fatty acid binding protein sample according to the first or second aspect as a sample, and the anti-L-FABP protein antibody as a reagent. Preferably, it further contains a labeled anti-L-FABP protein antibody, and if necessary, an adsorption inhibitor (BSA, etc.), a pretreatment solution (arbitrary buffer, optional surfactant, etc.), reaction buffer Liquid (arbitrary buffer etc.), chromogenic substrate (3,3 ′, 5,5′-tetramethylbenzidine, hydrogen peroxide solution etc.) and the like may be contained.
The L-FABP protein measurement kit is preferably a kit using a sandwich ELISA method in which two types of antibodies having different recognition sites for an antigen are used in combination, and any anti-L-FABP antibody on the solid phase and anti-labeled antibody can be used. More preferably, the kit uses L-FABP antibody clone 2.
Specific examples of the L-FABP protein measurement kit using the sandwich ELISA method include kits including the following (1) to (9).
(1) L-FABP antibody-immobilized microplate ... anti-human L-FABP mouse monoclonal antibody binding well (2) pretreatment solution (3) reaction buffer (4) enzyme-labeled antibody ... peroxidase-labeled anti-human L -FABP mouse monoclonal antibody [from clone 2 producing cell line]
(5) Enzyme substrate solution (6) Detergent (arbitrary buffer, surfactant, etc.)
(7) Reaction stop solution (1N sulfuric acid, etc.)
(8) Standard buffer (arbitrary buffer, etc.)
(9) Liver-type fatty acid binding protein preparation (9) As a liver-type fatty acid binding protein preparation, conventionally, a solution prepared by mixing recombinant human L-FABP with an arbitrary buffer has been used. A solution obtained by mixing the above-mentioned liver-type fatty acid binding protein preparation in a buffer solution is preferable, and the concentration of the preparation is not particularly limited, and examples thereof include 10 to 10000 ng / mL, preferably 50 to 5000 ng / mL, and 100 to 100 1000 ng / mL is more preferable, 200 to 800 ng / mL is still more preferable, and 300 to 600 ng / mL is particularly preferable.
L- using the sandwich ELISA method using the liver-type fatty acid-binding protein sample according to the first or second aspect, except that the conventional recombinant human L-FABP is used as the liver-type fatty acid-binding protein sample. As a commercially available kit similar to the FABP protein measurement kit, “Lenapro L-FABP Test TMB” (manufactured by Simic Holdings Co., Ltd.) can be mentioned.

The preservation solution of the liver-type fatty acid binding protein preparation comprising L-FABP protein is preferably a protein preservation buffer containing BSA for the purpose of preventing protein adsorption. For example, the following protein storage buffer is mentioned.
(Protein preservation buffer)
10 mM phosphate buffer (pH 7.2), 150 mM NaCl, 1.0% BSA, 0.1% NaN ₃

<Liver type fatty acid binding protein preparation with oxidation coefficient of variation set to 1.4 or less>
The liver-type fatty acid binding protein preparation according to the third aspect is subjected to an oxidation treatment at 25 ° C. for 1 hour with 10 mM oxidizing agent, and the oxidation treatment with respect to the measured value using the liver-type fatty acid binding protein preparation without oxidation treatment It includes a liver-type fatty acid binding protein having an oxidation variation coefficient represented by a ratio of measured values of 1.4 or less. When the oxidation variation coefficient is 1.4 or less, variation due to oxidation of the measured value can be suppressed.
Further, as another aspect of the liver type fatty acid binding protein preparation according to the third aspect, the liver type fatty acid binding protein comprising a liver type fatty acid binding protein consisting of an amino acid sequence having 90% or more identity with SEQ ID NO: 1 in the sequence listing, At least one fatty acid selected from the group consisting of arachidonic acid, oleic acid, 8-isoprostaglandin F _2α and 2,3-dinor-8-isoprostaglandin F _2α including.
The liver-type fatty acid binding protein sample according to the third aspect is preferably a liver-type fatty acid binding protein sample to be sold from the viewpoint of practical and commercial use.

In the present specification and claims, the oxidation variation coefficient is a liver type fatty acid binding protein preparation which is oxidized at room temperature (25 ° C.) for 1 hour with 10 mM oxidizing agent (for example, AAPH) and is not oxidized. The ratio of the measurement value using the liver-type fatty acid binding protein preparation with oxidation treatment to the measurement value using the above-mentioned oxidation is performed, and oxidation treatment is performed at room temperature (25 ° C.) for 1 hour with 10 mM AAPH, and the liver without oxidation treatment. Ratio of the measured value (for example, labeling intensity) using the liver-type fatty acid binding protein sample with oxidation treatment to the measured value using the type fatty acid binding protein sample (for example, the absorbance ratio (OD Ratio)).

OD value using the above-mentioned liver-type fatty acid binding protein preparation with oxidation treatment /
OD value using the above liver-type fatty acid binding protein preparation without oxidation treatment

In the liver type fatty acid binding protein preparation according to the third aspect, the oxidation variation coefficient is preferably 1.3 or less.
In the liver type fatty acid binding protein preparation according to the third aspect, the lower limit value of the oxidation variation coefficient is not particularly limited as long as the effect of the present invention is not impaired, but for example, 0.8 or more may be mentioned, and 0.9 It is preferable that it is above, and it is more preferable that it is 1.0 or more.

<Method for evaluating liver-type fatty acid binding protein preparation using oxidation coefficient of variation as an index>
The method for evaluating a liver-type fatty acid binding protein preparation according to the fourth aspect includes an oxidation represented by a ratio of a measurement value after the oxidation treatment to a measurement value using the liver-type fatty acid binding protein preparation before the oxidation treatment. Using the coefficient of variation as an index, the difficulty of oxidizing the sample is evaluated.
In the method for evaluating a liver-type fatty acid binding protein preparation according to the fourth aspect, the oxidation variation coefficient is preferably 1.4 or less, and preferably 1.3 or less, from the viewpoint of suppressing the oxidation variation range. More preferred.
The lower limit value of the oxidation variation coefficient is not particularly limited as long as the effect of the present invention is not impaired, and examples thereof include 0.8 or more, preferably 0.9 or more, and preferably 1.0 or more. More preferred.

<Method of suppressing fluctuation range of measured value in measurement using liver type fatty acid binding protein preparation>
The method for suppressing the fluctuation range of the measurement value caused by the liver-type fatty acid binding protein in the measurement using the liver-type fatty acid binding protein preparation according to the fifth aspect,
The oxidation rate of the 19th methionine of liver type fatty acid binding protein is 30% or more, or the oxidation rate of the 113th methionine is 70% or more, and arachidonic acid, oleic acid, 8-isoprostaglandin F _2α and At least one selected from the group consisting of containing at least one fatty acid selected from the group consisting of 2,3-dinor-8-isoprostaglandin F _{2α in the} liver type fatty acid binding protein preparation Including.
In the method according to the fifth aspect, the oxidation rate of the 19th methionine is preferably 35% or more, more preferably 38% or more, further preferably 40% or more, and 45% or more. It is particularly preferred that
Further, the oxidation rate of the 113th methionine is preferably 73% or more, more preferably 75% or more, and further preferably 80% or more.
In the method according to the fifth aspect, the content of the at least one fatty acid to be contained in the liver-type fatty acid binding protein preparation is not particularly limited as long as the effects of the present invention are not impaired. The molar amount of the protein is preferably 30 times or more, more preferably 50 times or more, even more preferably 75 times or more, and more preferably 100 times or more. It is particularly preferable that the molar amount is 300 times or more.
The upper limit of the fatty acid content is not particularly limited, but when the adsorption inhibitor is further contained, the fatty acid may be adsorbed to the adsorption inhibitor, so that the molar amount of liver-type fatty acid binding protein The above fatty acid can be contained in a molar amount of 500,000 times, preferably 200,000 times or less, more preferably 100,000 times or less, more preferably 10,000 times or less. Is more preferable, and a molar amount of 1000 times or less is particularly preferable.
According to the method of the fifth aspect, it is possible to suppress the fluctuation range of the measurement value in the measurement using the substance that specifically binds to the liver-type fatty acid binding protein, and preferably the fluctuation around 37 ° C. for 2 weeks. The width can be 15% or less, more preferably the fluctuation range can be 10% or less, still more preferably the fluctuation range can be 5% or less, and particularly preferably the fluctuation range can be 1% or less. it can.

<Method for preparing calibration curve and method for quantifying liver-type fatty acid binding protein>
The method for preparing a calibration curve for a liver-type fatty acid binding protein according to the sixth aspect uses the liver-type fatty acid binding protein preparation according to the first or second aspect.
Specifically, a calibration curve is created based on the relationship between the measured label intensity (eg, enzyme label intensity, fluorescence intensity, ultraviolet intensity, radiation intensity, etc.) and the amount (eg, concentration) of the standard. can do.
The method for quantifying liver-type fatty acid binding protein in the sample according to the seventh aspect uses the calibration curve created by the method according to the sixth aspect.
Specifically, under the same conditions as in the preparation of the calibration curve, the labeling strength of the liver-type fatty acid binding protein in the sample labeled with a labeled antibody or the like is measured, based on the calibration curve (for example, compared), It is possible to detect or quantify liver-type fatty acid binding protein in a sample.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further in detail, the scope of the present invention is not limited to these Examples.

Reference example 1
(Oxidation of methionine contained in human L-FABP protein)
FIG. 2 (a) shows the amino acid sequence of the human L-FABP protein shown in SEQ ID NO: 1, and FIG. 2 (b) shows various peptide fragments that can be generated by trypsin digestion of the human L-FABP protein. It is a figure which shows molecular weight.
MS spectra of various peptide fragments obtained by trypsin digestion of human L-FABP protein after reaction with AAPH at various concentrations (40 mM, 200 mM) were measured.
FIG. 3A shows peptide fragment No. 1 containing Met1 obtained by trypsin digestion of human L-FABP protein after reaction with AAPH at various concentrations (40 mM, 200 mM). FIG. 3 (b) is a peptide fragment No. 1 containing Met19 obtained by trypsin digesting the human L-FABP protein after the reaction with AAPH at various concentrations. FIG. 3 (c) is a peptide fragment No. 2 containing Met74 obtained by trypsin digestion of the human L-FABP protein after the reaction with the above-mentioned various concentrations of AAPH. 9 and peptide fragment no. FIG. 3 (d) shows the peptide fragment No. 10 containing Met113 obtained by trypsin digestion of the human L-FABP protein after reaction with AAPH at various concentrations. 14 shows MS spectra of 14 and peptide fragment 15. FIG.
From the results shown in FIGS. 3A to 3D, it is clear that the oxidation modification sites are the 19th, 74th, and 113th methionine residues (hereinafter also referred to as Met19, Met74, and Met113, respectively). became.

Reference example 2
(Method for producing mutant L-FABP protein)
A mutant protein obtained by mutating Met19 to a leucine residue (hereinafter also referred to as L-FABP M19L),
A mutant protein obtained by mutating Met74 to a leucine residue (hereinafter also referred to as L-FABP M74L),
A mutant protein obtained by mutating Met113 to a leucine residue (hereinafter also referred to as L-FABP M113L),
Mutant proteins obtained by mutating Met19 and Met74 to leucine residues (hereinafter also referred to as L-FABP M19L / M74L),
Mutant proteins obtained by mutating Met19 and Met113 to leucine residues (hereinafter also referred to as L-FABP M19L / M113L),
Mutant proteins obtained by mutating Met74 and Met113 to leucine residues (hereinafter also referred to as L-FABP M74L / M113L),
Mutant proteins (hereinafter also referred to as L-FABP M19L / M74L / M113L) were prepared by mutating three methionine residues (Met19, Met74, Met113) to leucine residues, respectively.
L-FABP M19L,
L-FABP M74L,
L-FABP M113L,
L-FABP M19L / M74L,
L-FABP M19L / M113L,
L-FABP M74L / M113L and L-FABP M19L / M74L / M113L are each expressed using a protein secretion expression system (CORYNEX (registered trademark): Ajinomoto Co., Inc.) using a Gram-positive bacterium Corynebacterium glutamicum. went.
The amino acid sequence of L-FABP M19L / M74L / M113L is shown in SEQ ID NO: 2 in the sequence listing below.
The gene sequence of L-FABP M19L / M74L / M113L used for protein expression is shown in SEQ ID NO: 3 in the Sequence Listing below.

The L-FABP M19L / M74L / M113L and the like which were expressed by the above-described method were first prepared using a centrifugal filter unit Amicon (registered trademark) Ultra-15, 3 kDa (manufactured by Millipore) and buffer A (10 mM Tris-HCl (pH 8)). 5), 1 mM DTT) and buffer exchange.
Next, purification was performed using a HiTrapQ FF column, 5 mL (manufactured by GE Healthcare). L-FABP M19L / M74L / M113L adsorbed on the HiTrapQ FF column was washed with buffer A, and then linear concentration gradient method using buffer A and buffer B (10 mM Tris-HCl (pH 8.5), 1 mM DTT, 2M NaCl). And fractions containing a peak at which buffer B was 3.1% were recovered. The recovered protein was subjected to protein staining with SDS-PAGE and Silver Stain II Kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.) to confirm the degree of purification.

  L-FABP M19L / M74L / M113L obtained by the above operation was stored at -80 ° C.

L-FABP protein solution (L-FABP M19L, L-FABP M74L, L-FABP M113L, L-FABP M19L / M74L) so that AAPH has a predetermined final concentration (0 mM, 0.5 mM, 1 mM, 2 mM, 4 mM), L-FABP M19L / M113L, L-FABP M74L / M113L, and L-FABP M19L / M74L / M113L solutions), reacted at room temperature for 1 hour, ELISA measurement was performed, and color development of labeled antibody (OD450 nm ) Compared to untreated samples. The comparison results are shown in FIG.
As is clear from the results shown in FIG. 4, it can be seen that the stability to oxidation is improved in L-FABP M19L and L-FABP M74L, and the oxidation rates of 19th and 113th methionine are dominant. confirmed. Further, it can be seen that L-FABP M19L / M74L / M113L is most excellent in stability against oxidation.
Next, L-FABP M19L / M74L / M113L, etc. whose oxidative stability was confirmed were dissolved in the above-mentioned protein storage buffer for long-term storage of the sample and stored at −80 ° C. Used for Examples 1-3 and 6.

Reference example 3
(Stability against oxidation)
AAPH was added to L-FABP M19L / M74L / M113L to a final concentration of 0 to 5 mM and reacted at room temperature for 1 hour. In consideration of the effect of air oxidation, an untreated sample in which only H ₂ O was added to the protein solution and measured immediately after the addition was used as a comparison target.

  These reaction solutions and untreated samples were subjected to ELISA measurement using “Lenapro L-FABP Test TMB” (manufactured by Simic Holdings Co., Ltd.), and the color development (OD 450 nm) of the labeled antibody was compared with the untreated sample. The diagnostic kit was used in accordance with the measurement method according to the attached package insert. The results are shown in FIG.

As is clear from the results of the ELISA measurement shown in FIG. 5, the L-FABP WT was confirmed to have an increase in the measured value of 112 to 121%, and the coefficient of variation (CV) representing the variation was 5.8%.
On the other hand, L-FABP M19L / M74L / M113L was observed only up to an increase of 102 to 107%, and CV was 2.4%.
From the above results, it was revealed that the antibody binding ability of L-FABP M19L / M74L / M113L was stable with respect to AAPH, and the fluctuation of the ELISA measurement value was small.
That is, it can be said that L-FABP M19L / M74L / M113L is stabilized against the oxidation reaction. In addition, even when placed at room temperature for 1 hour, the antibody-binding ability of L-FABP WT increases, but since L-FABP M19L / M74L / M113L does not increase, it can be said that it is stabilized against air oxidation.

Example 1
<Oxidation stability in ELISA measurement using labeled antibody with different recognition site from clone 2>
AAPH was added to the L-FABP protein solution at a predetermined final concentration (0 to 4 mM), reacted at room temperature for 1 hour, and ELISA measurement was performed. Clone 1, clone 2, and clone F were used as labeled antibodies, and the color development (OD 450 nm) was compared with the untreated sample. The results are shown in FIG.
As is clear from the results shown in FIG. 6, in the L-FABP measurement method using labeled antibodies from clone 1 and clone F, the antibody-binding ability of L-FABP WT is changed by oxidation as in clone 2, It can be seen that L-FABP M19L / M74L / M113L suppresses the change in antibody binding ability due to oxidation.
Therefore, the stabilized L-FABP protein is also effective in an ELISA measurement system using anti-L-FABP antibody clone 1 and anti-L-FABP antibody clone F having different antigen recognition sites.

ELISA measurement was performed in the same manner as in Example 1 by sandwich ELISA using the above anti-L-FABP antibody clone 2 as a labeled antibody, and the 19th, 74th and 113th of various L-FABP proteins having different oxidation progresses. The oxidation rate of methionine was measured. The results are shown in FIG.
In FIG. 7, * indicates that oxidization is approximately saturated and stabilized based on ELISA measurement data of WT L-FABP using clone 2 shown in Example 1 and FIG. 6 (various data with AAPH addition concentration). The range of the mean value ± 2SD (standard deviation) is the region where the “saturation region where the ELISA reactivity is increased by oxidation”.
As can be seen from FIG. 7, the minimum oxidation rate in the region where the oxidation is saturated and stabilized is about 38% for the 19th methionine, about 70% for the 74th methionine, and 113th. The methionine was about 73%.
From the above results, it can be said that the 19th methionine having an oxidation rate of 30% or more contributes to the reduction of the fluctuation range of the ELISA measurement value.
In addition, it can be said that the 113th methionine has an oxidation rate of 70% or more also contributes to a reduction in the fluctuation range of the ELISA measurement value.
From the results shown in FIG. 7, it can be expected that the increase in the ELISA measured value is saturated in the “saturation region where the ELISA reactivity increases due to oxidation” in FIG.

Example 2
<Stability at 37 ° C. of oxidized L-FABP sample>
L-FABP oxidized with 40 mM AAPH was stored at 37 ° C., and ELISA measurement was performed according to a conventional method using “Lenapro L-FABP Test TMB” every two weeks until 2 weeks later. Measurements were also performed on samples stored at 4 ° C. and used as comparison targets. Regarding the color development (OD450 nm) of the labeled antibody of the 37 ° C. storage sample, the ratio (%) where the color development (OD450 nm) of the labeled antibody of the 4 ° C. storage sample was 100 was compared. The results are shown in FIG.

As is clear from the results of the ELISA measurement shown in FIG. 8, the L-FABP subjected to the oxidation treatment was observed only up to 94 to 102%. On the other hand, an increase of 114 to 130% was observed for unoxidized L-FABP.
From the above results, it was revealed that the antibody-binding ability of L-FABP oxidized against long-term storage at 37 ° C. is stable, and the fluctuation of the ELISA measurement value is small. That is, it can be said that the L-FABP subjected to the oxidation treatment was stabilized even after long-term storage at 37 ° C.

Example 3
<Stability by adding fatty acid>
FIG. 9 (a) is a diagram showing changes in ELISA measurement values (rate (%) where 0 times the amount of fatty acid added is 100) due to the fatty acid addition treatment of the L-FABP protein.
It was revealed that the antibody binding ability of the L-FABP protein varies depending on the type of fatty acid to be bound (FIG. 9B) and the concentration.
FIG. 9 (a) shows the antibody-binding ability of 8-isoprostaglandin F _2α , 2,3-dinor-8-isoprostaglandin F _2α and entero-prostaglandin E ₂ to the L-FABP protein. Showing change. Arachidonic acid and oleic acid will be described later.

  Next, the final molar amounts of various fatty acids are added 200,000 times with respect to the molar amount of wild-type L-FABP protein (L-FABP WT) or L-FABP M19L / M74L / M113L. Reacted for hours. In consideration of the effect of air oxidation, an untreated sample in which only DMSO (final concentration 5%) was added to the protein solution and measurement was performed immediately after the addition was used as a comparative sample.

  These reaction solutions and untreated samples were subjected to ELISA measurement using “Lenapro L-FABP Test TMB” in the presence of 1% by mass of BSA, and the color development (OD450 nm) of the labeled antibody was compared with the untreated sample. The diagnostic kit was used in accordance with the measurement method according to the attached package insert. The results are shown in FIGS. 10 (a) to (c).

FIG. 10 (a) is a diagram showing changes in ELISA measured values due to addition of various concentrations of arachidonic acid, and FIG. 10 (b) is a diagram showing changes in ELISA measured values due to addition of various concentrations of oleic acid. (c) is a graph showing changes in ELISA measurements by 8 iso static prostaglandin _{F 2.alpha} addition of various concentrations.
As is clear from the results of the ELISA measurement shown in FIGS. 10 (a) to (c), L-FABP WT showed an increase in the measured value of 104 to 220% for arachidonic acid (CV 34.0%), and for oleic acid. Increased 100-124% (CV 10.4%), and 8-isoprostaglandin F _2α was confirmed to have a measured value variation of 95-118% (CV 6.8%).
On the other hand, L-FABP M19L / M74L / M113L has a measured value variation of 88 to 103% for arachidonic acid (CV 6.1%), a measured value variation of 92 to 107% for oleic acid, (CV 5.1%) ), An increase in the measured value of 96 to 101% (CV 2.3%) was confirmed for 8-isoprostaglandin F _2α .
From the results shown in FIG. 10, as the fatty acid is added to L-FABP WT, the change in the ELISA measurement value approaches saturation, and a liver type fatty acid binding protein containing a specific amount or more of fatty acid is used as a sample. This shows that the fluctuation range of the ELISA measurement value can be suppressed.
In particular, from the results shown in FIG. 10 (a), it can be seen that when arachidonic acid is included, the change in the ELISA measurement value approaches saturation with a relatively small molar amount.
In addition, it is estimated that at least a part of the added fatty acid is adsorbed to BSA without binding to L-FABP WT.
Moreover, it became clear that the antibody-binding ability of L-FABP M19L / M74L / M113L is stable with respect to the addition of fatty acid, and the fluctuation of the ELISA measurement value is small. That is, it can be said that L-FABP M19L / M74L / M113L was stabilized against fatty acid addition.

Example 4
<Suppression of fluctuation in ELISA measurement value by binding fatty acid (arachidonic acid or oleic acid) to L-FABP>
FIG. 11 and FIG. 12 are diagrams showing the amount of fatty acid (arachidonic acid or oleic acid) added and the change in the ELISA measurement value (ratio (%) where the amount of fatty acid added is 0 mol times 100).
In the absence of BSA, 0 to 100 moles of fatty acid (arachidonic acid or oleic acid) was added to L-FABP, reacted at room temperature for 60 minutes, and then dialyzed overnight against physiological saline. did. On the next day, the dialysis external solution was changed and dialysis was again performed overnight to remove free fatty acids not bound to L-FABP. FIGS. 11 and 12 show changes in the ELISA measurement values of the obtained samples (ratio (%) where the amount of fatty acid added is 0 mol times 100).
As is clear from FIG. 11, the change in the ELISA measurement value due to the addition of arachidonic acid became constant when 10 mole times or more of fatty acid was added to L-FABP, and 30 moles or more of arachidonic acid was added. It was confirmed that it became constant.
In addition, as is clear from FIG. 12, the change in the ELISA measurement value due to the addition of oleic acid becomes constant when adding 100 mol times or more of fatty acid to L-FABP, and 300 mol times or more of oleic acid is added. It was confirmed that it became constant when added.

Example 5
<Stability at 37 ° C. of L-FABP preparation conjugated with arachidonic acid or oleic acid>
Using a sample prepared by diluting L-FABP bound with a molar amount of arachidonic acid containing 50 times the molar amount of L-FABP in the same manner as in Example 4 in a BSA-containing protein storage buffer Then, the sample was stored in the same manner as in Example 3, and the change in ELISA measured values at 37 ° C. for 2 weeks was confirmed. The results are shown in FIG.
Similarly, by the same method using a sample prepared by diluting L-FABP combined with oleic acid containing 1000 times the molar amount of L-FABP in a BSA-containing protein storage buffer. The change of the ELISA measured value at 37 ° C. for 2 weeks was confirmed. The results are shown in FIG.

As is clear from the results of the ELISA measurement shown in FIG. 13, only 99.9 to 100.3% fluctuation was observed in L-FABP to which arachidonic acid was bound. On the other hand, L-FABP not bound with arachidonic acid was observed to increase by 118 to 132%.
From the above results, it was revealed that the antibody binding ability of L-FABP to which arachidonic acid was bound was stable for long-term storage at 37 ° C., and the fluctuation of the ELISA measurement value was small.
Similarly, as is clear from the results of the ELISA measurement shown in FIG. 14, the fluctuation of L-FABP bound to oleic acid was only 100 to 110%. On the other hand, an increase of 120 to 130% was observed for L-FABP not bound with oleic acid.
From the above results, it was revealed that the antibody binding ability of L-FABP to which oleic acid was bound was stable for long-term storage at 37 ° C., and the fluctuation of the ELISA measurement value was small.

Example 6
<Evaluation of L-FABP sample using oxidation coefficient of variation>

L-FABP M19L / M74L / M113L, L-FABP WT treated with 0.5 mM AAPH, L-treated with 4 mM AAPH, shown in Example 1 and FIG. FABP WT and L-FABP WT were oxidized with 10 mM AAPH at 25 ° C. for 1 hour, and the oxidation variation coefficient was calculated from the OD ratio of the ELISA measurement values with and without the oxidation treatment. As a control, L-FABP WT was also calculated. The results are shown in FIG.
As is clear from the results shown in FIG. 15, the oxidation variation coefficient of L-FABP WT as a control was about 1.8.
On the other hand, L-FABP M19L / M74L / M113L, which was shown to have a small variation in ELISA measurement values in Example 1 and FIG. 6, L-FABP WT treated with 0.5 mM AAPH, L treated with 4 mM AAPH -FABP WT all had an oxidation variation coefficient of 1.3 or less.
Therefore, L-FABP M19L / M74L / M113L, L-FABP WT treated with 0.5 mM AAPH, average value of oxidation fluctuation coefficient of L-FABP WT treated with 4 mM AAPH + 2SD (standard deviation). If it is 4 (1.2 + 0.2) or less, it can be said that the fluctuation of the measured value is suppressed with respect to oxidation, and that it is stable.

Next, each of various L-FABP proteins having different oxidation progresses used in Example 1 and FIG. 7 was oxidized with 10 mM AAPH at 25 ° C. for 1 hour, and the OD of the ELISA measurement value with or without the oxidation treatment was performed. The oxidation variation coefficient was calculated from the ratio. Thereafter, for each of the 19th, 74th, and 113th methionines, the correlation between the methionine oxidation rate and the oxidation variation coefficient was determined as follows: the residual unoxidized methionine content (100% -methionine oxidation rate) and the oxidation variation coefficient; The correlation was calculated. The results are shown in FIG.
As is clear from the results shown in FIG. 16, for the 19th methionine, the oxidation variation coefficient converged when the residual unoxidized methionine content was less than 70% (that is, the oxidation rate was 30% or more). It can be seen that oxidation fluctuation can be suppressed.
In particular, it can be said that suppression of oxidation fluctuation is particularly excellent when the residual unoxidized methionine content is less than 62% (that is, the oxidation ratio is 38% or more), which is a case where the oxidation fluctuation coefficient is 1.4 or less.

In addition, regarding the 74th methionine, it can be seen that when the residual unoxidized methionine content is less than 30% (that is, the oxidation rate is 70% or more), the oxidation variation coefficient converges and the oxidation variation is suppressed.
In particular, it can be said that the suppression of the oxidation fluctuation is particularly excellent when the residual unoxidized methionine content is less than 25% (that is, the oxidation ratio is 75% or more), which is a case where the oxidation fluctuation coefficient is 1.4 or less.
In addition, regarding the 113th methionine, it can be seen that when the residual unoxidized methionine content is less than 30% (that is, the oxidation rate is 70% or more), the oxidation variation coefficient converges and the oxidation variation is suppressed.

Also, (1) L-FABP WT, (2) oleic acid-containing L-FABP in Examples 4 and 5, (3) arachidonic acid-containing L-FABP, (4) L-FABP treated with 0.5 mM AAPH The coefficient of oxidation variation was calculated for WT, (5) L-FABP WT treated with 4 mM AAPH, and (6) L-FABP M19L / M74L / M113L. The calculation results are shown in FIG.
As is clear from the results shown in FIG. 17, L-FABP WT has an oxidation variation coefficient greatly exceeding 1.4 (about 1.8) and does not satisfy the stability against oxidation as an L-FABP standard. I understand.
On the other hand, (2) oleic acid-containing L-FABP, (3) arachidonic acid-containing L-FABP, (4) L-FABP WT treated with 0.5 mM AAPH, (5) L-FABP treated with 4 mM AAPH It can be seen that WT and (6) L-FABP M19L / M74L / M113L both have an oxidation variation coefficient of 1.4 or less and are excellent in stability against oxidation.

SEQ ID NO: 1: Amino acid sequence of L-FABP WT SEQ ID NO: 2: Amino acid sequence of L-FABP M19L / M74L / M113L SEQ ID NO: 3: DNA sequence of L-FABP M19L / M74L / M113L

Claims (7)

  It consists of an amino acid sequence having 90% or more identity with SEQ ID NO: 1 in the Sequence Listing, and the 19th methionine has an oxidation rate of 30% or more, or the 19th methionine has an oxidation rate of 30% or more and the 113th A liver type fatty acid binding protein preparation containing a liver type fatty acid binding protein having an oxidation rate of 70% or more of methionine.   The liver-type fatty acid-binding protein preparation according to claim 1, wherein the measured value in the measurement using a substance that specifically binds to the liver-type fatty acid-binding protein has a fluctuation range of about 10% or less at 37 ° C for about 2 weeks.   The liver type fatty acid binding protein sample according to claim 1 or 2, which is offered for sale.   The liver-type fatty acid binding protein preparation according to any one of claims 1 to 3, which is used as a standard substance for measurement or a substance for quality control. A method for suppressing a fluctuation range of a measurement value in a measurement using a liver type fatty acid binding protein preparation,
The oxidation rate of the 19th methionine of the liver fatty acid binding protein consisting of an amino acid sequence having 90% or more identity with SEQ ID NO: 1 in the sequence listing is set to 30% or more, or the oxidation rate of the 19th methionine is set to 30%. The method including the above and the oxidation rate of the 113th methionine being 70% or more.   A method for preparing a calibration curve for a liver-type fatty acid binding protein using the liver-type fatty acid binding protein preparation according to any one of claims 1 to 4. A method for quantifying liver-type fatty acid binding protein in a sample using the calibration curve prepared by the method according to claim 6.