JPWO2017217514A1 - Liver-type fatty acid binding protein preparation, method for evaluating the preparation, method for suppressing fluctuation of measurement value caused by liver-type fatty acid binding protein in measurement using the preparation, liver-type fatty acid binding protein, the protein DNA encoding, cell transformed with the DNA, method for producing the protein, method for preparing a calibration curve of liver type fatty acid binding protein, and method for quantifying the protein - Google Patents

Abstract

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 specifically binding substance, a method for evaluating the preparation, a liver-type fatty acid binding protein Providing a method of preparing a calibration curve and a method of quantifying the protein. The oxidation variation coefficient represented by the ratio of the measured value obtained by the oxidation treatment to the measured value using a liver-type fatty acid binding protein preparation not oxidized at 25 ° C. for 1 hour with 10 mM oxidizing agent is 1.4 or less A liver-type fatty acid binding protein preparation set for the invention, a liver-type fatty acid binding protein used for the preparation, a DNA encoding the protein, a cell transformed with the DNA, a method for producing the protein, the preparation A method of evaluating, a method of suppressing fluctuation of measured value in measurement using the preparation, a method of preparing a calibration curve of the protein, and a method of quantifying the protein.

Description

  The present invention provides 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 specifically binding substance, a method for evaluating the preparation, and the standard For suppressing the fluctuation range of the measurement value caused by liver type fatty acid binding protein in the measurement using the product, liver type fatty acid binding protein, DNA encoding said protein, cells transformed with said DNA, method for producing said protein The present invention relates to a method of preparing a calibration curve of a liver-type fatty acid binding protein, and a method of quantifying the protein.

A fatty acid binding protein (Fatty Acid-Binding Protein; hereinafter also referred to as FABP) is a protein with a molecular weight of about 14 kDa that belongs to the intracellular lipid binding protein family, and reversibly binds to a hydrophobic ligand such as fatty acid to carry out intracellular transport. It is known that it bears (for example, nonpatent literature 1). Among them, liver-type 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 tubular cells of the liver and kidney, and urine Urinary excretion is increased in response to ischemia / oxidative stress due to tubular injury (for example, Non-Patent Document 2). Therefore, examination of a renal disease is possible by detecting kidney tissue origin L-FABP protein in urine (for example, patent documents 1).
Also, as shown in FIG. 24, the L-FABP protein is stabilized in such a manner that two α-helices cover a β-barrel structure in which two anti-parallel β-sheets are orthogonal to each other. It is known to bind to free fatty acids (eg, oleic acid) (PDB ID: 2 LKK) (Non-patent Document 3).
And L-FABP protein transports free fatty acid to mitochondria and peroxisome, and has a mechanism which promotes beta oxidation (nonpatent literature 4).
As the binding affinity of fatty acid ligands, the carbon chains of fatty acids are elongated, and the double bond tends to increase as the number of double bonds increases (Non-patent Documents 5 and 6), and L-FABP proteins are particularly suitable for peroxides. It has been reported that the binding affinity is high (Non-patent Document 7).
Moreover, in the report which studied the antioxidant mechanism of L-FABP protein, it is shown that the methionine residue of rat L-FABP protein was oxidized by AAPH (nonpatent literature 8).

Japanese Patent Application Publication No. 11-242026

Furuhashi, M .; , Et al. : Nat Rev Drug Discov, 7: 489-503, 2008 Kamijo, A. et al. et al. : J Lab Clin Med, 143: 23-30, 2004 Cai, J. et al. 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. et al. W. et al. : Int J Biochem Cell Biol, 33: 865-876, 2001 Norris, A .; W. Spector, A., et al. A. J Lipid Res, 43: 646-653, 2002. Raza, H. et al. : Biochem Biophys Res Commun, 161: 448-455, 1989 Yan, J. et al. et al. J Lipid Res, 50: 2445-2454, 2009

As a test kit for detecting L-FABP protein as described above, for example, a test kit employing a sandwich ELISA method using two types of antibodies having different recognition sites for L-FABP protein has been developed. .
Here, FIG. 1 is a view showing the storage stability of L-FABP, and the graph in FIG. 1 shows changes in ELISA measurement values according to the number of storage days when L-FABP is stored at 4 ° C., 25 ° C. or 37 ° C. (The ratio (%) when making a -80 degreeC storage sample into 100) is shown.
In the above test kit employing the sandwich ELISA method, when the conventional L-FABP protein is stored for a long time at room temperature (25 ° C.) or more with the conventional measurement standard substance (standard product) using the recombinant L-FABP protein, FIG. As shown, there is a problem that the antibody binding ability is changed, and accurate measurement of L-FABP protein can not be performed by an immunological method utilizing an antigen-antibody reaction such as ELISA.
Thus, 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 low-temperature storage is required for the preparation, and further operability and stability are required. Improvement was desired.

  The present invention has been made in view of the above-mentioned 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 specifically binding substance A method for evaluating the preparation, a method for suppressing the fluctuation 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 of the liver-type fatty acid binding protein, and the protein Aims to provide a method to quantify

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that L-FABP protein is a methionine residue by treatment with an oxidizing agent 2,2'-azobis 2-amidinopropane (hereinafter abbreviated as AAPH). It has been found that oxidative modification and structural change occur in the antibody, and as a result, the antibody binding ability in the ELISA is changed, and the ELISA measurement value largely fluctuates. In addition, it was also surprisingly found that, even in the case of no addition of AAPH (addition of H ₂ O), the ELISA measurement value was raised by reacting at room temperature for 1 hour, and the effect of air oxidation was caused. . In particular, it was found that among the 19th, 74th and 113th methionines, the oxidation rates of the 19th and 113th methionines are dominant in suppressing the fluctuation range of the ELISA measurement value.

Furthermore, L-FABP protein in which the methionine residue in L-FABP protein is mutated to another amino acid by genetic modification technology changes antibody binding ability even in oxidation reaction, addition of fatty acid, and long-term storage at room temperature or more It was found to 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
It is a liver-type fatty acid binding protein preparation whose oxidation variation coefficient is set to 1.4 or less.
The second aspect of the present invention is
A liver-type fatty acid binding protein consisting of an amino acid sequence having 90% or more identity to SEQ ID NO: 1 in the sequence listing, and one or more methionines at 19th, 74th and 113th being substituted with nonpolar amino acids other than methionine It is a liver-type fatty acid binding protein preparation, which comprises a liver-type fatty acid binding protein in which at least the 19th methionine is substituted with a nonpolar amino acid other than methionine.
The third aspect of the present invention is
The amino acid sequence having an identity of 90% or more with SEQ ID NO: 1 of the sequence listing, which is used for the preparation of a liver-type fatty acid binding protein preparation according to the first aspect, and one or more methionine of 19th, 74th and 113th Is a liver-type fatty acid binding protein substituted with a nonpolar amino acid other than methionine.
The fourth aspect of the present invention is
It is a DNA encoding the protein according to the third aspect.
The fifth aspect of the present invention is
It is a cell transformed with the DNA according to the fourth aspect.
The sixth aspect of the present invention is
A method for producing a protein, comprising the steps of culturing the cell according to the fifth aspect and recovering the protein according to the third aspect.

The seventh aspect of the present invention is
A liver-type fatty acid binding protein comprising an amino acid sequence having an identity of 90% or more with SEQ ID NO: 1 in the sequence listing and having an oxidation rate of 30% or more at the 19th methionine or 70% or more of the 113th methionine It is a liver-type fatty acid binding protein preparation containing.
The eighth aspect of the present invention is
At least the oxidation coefficient of variation is selected from 1.4 to qs with arachidonic acid, oleic acid, 8 iso static Prostaglandin F _2.alpha and 2,3-dinor-8-iso static prostaglandin F group consisting _2.alpha A liver-type fatty acid binding protein preparation comprising a liver-type fatty acid binding protein comprising one type of fatty acid and consisting of an amino acid sequence having an identity of 90% or more with SEQ ID NO: 1 in the sequence listing.

The ninth aspect of the present invention is
It is a method of evaluating a liver-type fatty acid binding protein preparation using an oxidation variation coefficient as an index.
The tenth aspect of the present invention is
A method for suppressing the fluctuation range of measurement values in measurement using a liver-type fatty acid binding protein preparation,
(1) At least one methionine in the 19th, 74th and 113th positions of the liver-type fatty acid binding protein consisting of an amino acid sequence of 90% or more identity to SEQ ID NO: 1 in the sequence listing is substituted with nonpolar amino acids other than methionine And replacing at least the 19th methionine with a nonpolar amino acid other than methionine,
(2) The oxidation rate of methionine of the 19th methionine of the liver-type fatty acid binding protein consisting of an amino acid sequence of 90% or more identity to SEQ ID NO: 1 in the sequence listing is 30% or more, or the oxidation rate of methionine of the 113th is 70% or more And (3) at least one fatty acid selected from the group consisting of arachidonic acid, oleic acid, 8-isostataglandin F _2α and 2,3-dinor-8-isoprostaglandin F _2α It is a method including at least one selected from the group consisting of including the above-mentioned liver-type fatty acid binding protein preparation.
An eleventh aspect of the present invention is
It is a method of preparing a calibration curve of a liver-type fatty acid binding protein using the liver-type fatty acid binding protein preparation according to any one of the first, second, seventh and eighth aspects.
The twelfth aspect of the present invention is
It is a method of quantifying a liver-type fatty acid binding protein in a sample using the calibration curve prepared by the method according to the eleventh aspect.

According to the present invention, a standard of liver-type fatty acid binding protein preparation, liver-type fatty acid binding protein, which can reduce the fluctuation range of the measurement value caused by liver-type fatty acid binding protein in measurement using a specifically binding substance It is possible to provide a method of producing and a method of quantifying the protein.
The liver-type fatty acid binding protein preparation of the present invention can suppress the change in antibody binding ability due to oxidation or fatty acid binding state, so it is not based on the derived 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.
In addition, 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 changes due to oxidation as a coefficient.

The liver-type fatty acid binding protein preparation of the present invention can reduce the fluctuation range of the measurement value due to the liver-type fatty acid binding protein in the measurement using a substance (for example, an antibody) that specifically binds. It 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 due to the liver-type fatty acid binding protein in the measurement using a substance that specifically binds, it is possible to measure standard substances or It is expected that production control of the hepatic fatty acid binding protein as a quality control substance is facilitated, and operability and versatility of measurement in the immunological method are improved.

It is a figure which shows the storage stability of L-FABP protein. (A) is a figure which shows the molecular weight measurement result by LC-ESI-MS of oxidized L-FABP and nonoxidized L-FABP, (b) is the fluorescence of oxidized L-FABP and nonoxidized L-FABP It is a figure which shows a spectrum. It is a figure which shows the change (The ratio (%) at the time of making untreated a 100) of the ELISA measured value by AAPH process of L-FABP protein. It is a figure which shows MS spectrum after adding various concentrations of AAPH to human L-FABP protein, and making it react at 37 degreeC for 1.5 hours. (A) shows the amino acid sequence of human L-FABP protein shown in SEQ ID NO: 1, (b) shows the estimated molecular weight of various peptide fragments which can be produced by trypsin digestion of human L-FABP protein It is. (A) is a peptide fragment No. 1 containing Met1 obtained by trypsin digestion of human L-FABP protein after reaction with the above-mentioned various concentrations (40 mM, 200 mM) of AAPH. 1 (b) shows a peptide fragment No. 1 containing Met19 obtained by trypsin digestion of human L-FABP protein after reaction with various concentrations of AAPH. FIG. 10 (c) is a diagram showing a Met74-containing peptide fragment No. 1 obtained by trypsin digestion of human L-FABP protein after reaction with the above-mentioned various concentrations of AAPH. 9 and peptide fragment no. 10 shows MS spectra of 10, and (d) is peptide fragment No. 1 containing Met113 obtained by trypsin digestion of human L-FABP protein after reaction with various concentrations of AAPH. 14 shows MS spectra of 14 and peptide fragment 15. FIG. FIG. 6 shows L-FABP M19L / M74L / M113L and purified protein expressed by CORYNEX®. It is a figure which shows the change (The ratio (%) when the case of non-processing is made into 100) of the ELISA measured value of the mutation L-FABP protein by AAPH treatment of various density | concentrations. It is a figure which shows the change (The ratio (%) which made the untreated sample 100) by the AAPH process of mutant | variant L-FABP protein. It is a figure which shows the change (The ratio (%) when making a -80 degreeC storage sample into 100) of the ELISA measured value by room temperature long-term storage (25 degreeC) of mutant L-FABP protein. It is a figure which shows the change (The ratio (%) made into 100 of -80 degreeC storage sample) by 37 degreeC extended storage of mutant L-FABP protein. Clone 2 is a figure which shows the influence (the ratio (%) which made the untreated sample 100) in ELISA measurement using the labeled antibody from which a recognition site differs. It is the figure which measured the oxidation rate of each 19th, 74th, and 113th methionine of various L-FABP protein in which progress of oxidation differs. It is a figure which shows the change (The ratio (%) on the 4 degreeC storage sample 100) by the 37 degreeC 2 week storage of the oxidized L-FABP protein. (A) is a figure which shows the change (The ratio (%) which made the fatty-acid addition amount 0 molar amount 100 100) by the various fatty acids of various density | concentrations. (B) is a figure which shows the kind of various fatty acids. It is a figure which shows the change (The ratio (%) which made the untreated sample 100) by the fatty acid addition process of L-FABP mutein. After addition of arachidonic acid in the absence of BSA, change in arachidonic acid addition amount and ELISA measurement value when free arachidonic acid was removed by dialysis (ratio (%) based on 0 molar volume of arachidonic acid addition amount as 100) FIG. After addition of oleic acid in the absence of BSA, changes in the addition amount of oleic acid and the ELISA measurement value when free oleic acid was removed by dialysis (ratio (%) based on 0 molar amount of oleic acid added as 100) FIG. After 50 molar volumes of arachidonic acid were added in the absence of BSA, the free arachidonic acid was removed by dialysis and L-FABP protein bound to arachidonic acid was changed in ELISA measurement value by two-week storage at 37 ° C (4 ° C It is a figure which shows the ratio (%) which made the preservation | save sample 100. FIG. After addition of 1000 molar volumes of oleic acid in the absence of BSA, dialysis removes the free oleic acid and changes the ELISA measurement value by 2 weeks storage at 37 ° C. of oleic acid-bound L-FABP protein (4 ° C. It is a figure which shows the ratio (%) which made the preservation | save sample 100. FIG. FIG. 6 shows the oxidation variation coefficient for L-FABP WT treated with L-FABP M19 L / M 74 L / M 113 L, 0.5 mM or 4 mM of AAPH, and L-FABP WT. It is a figure which shows correlation with the content rate of a residual unoxidized type methionine, and an oxidation variation coefficient about each of the 19th, 74th, and 113th methionine. It is a figure which shows the calculation result of the oxidation variation 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, but the present invention is not limited to the following embodiments at all, and can be implemented with appropriate modifications within the scope of the object of the present invention. .

«Liver-type fatty acid binding protein preparation»
In the present specification, “standard product” refers to a standard substance for measurement (calibrator) and a substance for quality control (control) as a typical example, and is a standard reference substance for practical use, a practical standard substance, a calibration substance for manufacturer's product, a standard for diagnosis. A substance, all standard products such as calibration standard substances, and other substances including quality control substances etc. are also included (Giaki Iizuka et al., “Current Status of Traceability Chain and Uncertainty” Biological Sample Analysis Vol. 34 , No. 3 (2011), pp. 179-188, standard products and standard substances in the Japanese Pharmacopoeia, 2009 JCCLS (Japan Clinical Laboratory Standards Council) Terminology Committee Terms (English) and their translated Japanese words (draft) 226).

Liver-type fatty acid binding protein preparation whose oxidation variation coefficient is set to 1.4 or less
The liver-type fatty acid binding protein preparation according to the first aspect is subjected to an oxidation treatment at 25 ° C. for 1 hour with 10 mM oxidizing agent, and the oxidation treatment to the measured value using a liver-type fatty acid binding protein preparation without oxidation treatment It includes a liver-type fatty acid binding protein whose oxidation variation coefficient, which is represented by the ratio of measured values, is set to 1.4 or less. By the oxidation variation coefficient being 1.4 or less, the variation due to oxidation of the measured value can be suppressed.
The liver-type fatty acid binding protein preparation according to the first aspect is preferably a liver-type fatty acid binding protein preparation to be sold, from the viewpoint of practical and commercial use.

In the present specification and claims, the oxidation variation coefficient refers to a liver-type fatty acid-binding protein preparation that is subjected to oxidation treatment at room temperature (25 ° C.) for 1 hour with 10 mM of an oxidizing agent (eg, AAPH) Ratio of the measured value using the above-mentioned liver-type fatty acid binding protein preparation with oxidation treatment to the measurement value using the above-mentioned, and the oxidation treatment without oxidation treatment by performing oxidation treatment at room temperature (25 ° C) for 1 hour with 10 mM AAPH. Ratio of the measured value (for example, labeling intensity) using the above-mentioned oxidation-treated liver-type fatty acid binding protein preparation to the measurement value using the fatty acid-binding protein preparation (for example, the absorbance ratio (OD Ratio)) is preferred.

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

In the liver-type fatty acid binding protein preparation according to the first aspect, the oxidation variation coefficient is preferably 1.3 or less.
In the liver-type fatty acid binding protein preparation according to the first 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, and for example, 0.8 or more can be mentioned. It is preferable that it is the above, and it is more preferable that it is 1.0 or more.

(The liver-type fatty acid binding protein preparation according to the second aspect)
The liver-type fatty acid binding protein preparation according to the second aspect comprises an amino acid sequence having an identity of 90% or more with SEQ ID NO: 1 in the sequence listing, and one or more methionine of 19th, 74th and 113th is methionine It comprises a mutant liver-type fatty acid binding protein according to the following third aspect, which is substituted with a nonpolar amino acid other than the above. By containing the following mutant liver-type fatty acid binding proteins, it is possible to suppress the fluctuation of binding ability due to the specifically binding substance.
In the liver-type fatty acid binding protein preparation according to the second aspect, the oxidation fluctuation coefficient is set to 1.4 or less even if the liver-type fatty acid binding protein preparation is set to an oxidation fluctuation coefficient of 1.4 or less It may not be a liver-type fatty acid binding protein preparation, but is preferably a liver-type fatty acid binding protein preparation whose oxidation variation coefficient is set to 1.4 or less.

(Mutated liver-type fatty acid binding protein)
Even if the mutant liver-type fatty acid binding protein according to the third aspect is a protein used for the liver-type fatty acid binding protein preparation according to the first aspect, the liver-type fatty acid binding protein preparation according to the first aspect It does not have to be the protein used,
It is preferable that it consists of an amino acid sequence having 90% or more identity with SEQ ID NO: 1 in the Sequence Listing, and 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 according to the third aspect is specific to a liver-type fatty acid-binding protein by replacing one or more of the 19th, the 74th, and the 113th methionine with a nonpolar amino acid other than methionine. The binding ability of a substance (eg, an antibody) that binds to
Besides methionine, oxidation modification such as cysteine residue which receives direct addition of disulfide bond or oxygen, lysine residue to be carbonylated, arginine residue, proline residue, tyrosine residue to be nitrated is known. (Toda T., et al., 35 (3); 17-22, 2011), 19th, 74th, 113th amino acids replacing one or more methionines are nonpolar amino acids Can prevent oxidative modification.

SEQ ID NO: 1 represents the amino acid sequence of a wild-type human L-FABP protein (hereinafter also referred to as L-FABP WT).
The term "amino acid sequence of 90% or more of identity" as used herein means that the identity of amino acids is 90% or more, preferably 95% or more, more preferably 97% or more. .
Even if it is a mutant protein by substitution, insertion, deletion, addition, etc. on the amino acid sequence of the wild type human liver-type fatty acid binding protein described in SEQ ID NO: 1 in the sequence listing, the mutation is a wild-type human liver-type fatty acid binding protein These mutations can all be within the range of liver-type fatty acid binding proteins if they are highly conserved in the three-dimensional structure of Specifically, one or more amino acids (for example, histidine (His)) at at least one end selected from the group consisting of the N-terminus and the C-terminus of an amino acid sequence having 90% or more identity to SEQ ID NO: 1 in the sequence listing. And alanine (Ala etc.), and even if the mutant protein is less than 90% identical (for example, 85% or more identical) to SEQ ID NO: 1 in the sequence listing, the mutation is wild type human liver type If the mutations are highly conserved in the three-dimensional structure of the fatty acid binding protein, they can all fall within the range of liver-type fatty acid binding protein.
The side chains of amino acids that are constituents of proteins differ from each other in hydrophobicity, charge, size, etc., meaning 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 highly conserved relationships are known empirically and by physical and chemical 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-type fatty acid binding protein is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by genetic recombination technology.
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 a suitable cDNA library or the like and performing gene recombination using this.

  In the mutant liver-type fatty acid binding protein according to the third aspect, it is preferable that the 19th, the 74th, and the 113th two or more methionines be replaced with nonpolar amino acids other than methionine, and the 19th methionine It is more preferable that one or more methionines be substituted with nonpolar amino acids other than methionine, and it is even more preferable that all of the 19th, 74th and 113th methionines be substituted with nonpolar amino acids other than methionine.

  As nonpolar amino acids that substitute one or more methionines at the 19th, 74th, and 113th positions, one or more methionine at the 19th, 74th, and 113th positions may be substituted with the same nonpolar amino acid, It may be substituted with different nonpolar amino acids. The nonpolar amino acids substituting one or more of the 19th, 74th and 113th methionines are preferably leucine, isoleucine, valine, alanine, phenylalanine and tryptophan, and methionine which does not significantly change the antibody binding property From the viewpoint of the 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-type fatty acid binding protein)
The DNA according to the fourth aspect is a DNA encoding the above mutant liver-type fatty acid binding protein.
The DNA (mutant gene) encoding the above mutant liver-type 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 L-FABP protein have been already reported, for example, primers are designed based on them, and cDNA is cloned from a suitable cDNA library etc. by PCR method, It can be obtained by genetic recombination. Site-directed mutagenesis, which is one of the genetic engineering techniques, is useful because it is a technique that can introduce a specific mutation at a specific position, and is useful in molecular cloning, 2nd edition, Current Protocols in Molecular. It can carry out according to the method as described in the biology etc.

(Transformed cells)
The cell according to the fifth aspect is a cell transformed with the DNA according to the fourth aspect.
A transformed cell can be prepared by introducing the DNA according to the fourth aspect or the recombinant vector containing the DNA according to the fourth aspect into a suitable host.
The recombinant vector containing the above DNA can be prepared by a general genetic recombination technique using an appropriate host vector system. As a suitable vector, a plasmid derived from E. coli (eg, pBR322, pUC118 etc.), a plasmid derived from Bacillus subtilis (eg, pUB110, pSH19 etc.), and further, animal viruses such as bacteriophage, retrovirus and vaccinia virus can be used. .
Host cells into which the above DNA or a recombinant vector containing the above DNA is introduced include bacteria, yeast and the like.
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. Can be mentioned. Transformation of these bacteria may be carried out by using a competent cell by a protoplast method or a known method.
Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, and examples thereof include Saccharomyces cerevisae (Saccharomyces cerevislae) and Saccharomyces kluyveri. Examples of the method for introducing the recombinant vector into the yeast host include electroporation, spheroplast method, lithium acetate method and the like.
As the cells transformed with the above DNA, Corynebacterium glutamicum can be used because the above mutant liver type fatty acid binding protein is efficiently produced and purification of the liver type fatty acid binding protein described later does not require complicated steps. It is preferable that it is a protein secretion expression system (CORYNEX (registered trademark): manufactured by Ajinomoto Co., Ltd.).

(Method for producing the above-mentioned mutant liver-type fatty acid binding protein)
The method for producing the mutant liver-type fatty acid binding protein according to the sixth aspect preferably includes the step of culturing the transformed cell and recovering the protein.
The transformed cells are cultured in an appropriate nutrient medium under conditions that allow expression of the introduced gene. In order to recover the above-mentioned protein from the culture of the above-mentioned transformed cells, a conventional isolation and purification method of protein may be used.
For example, when the above protein is expressed in a dissolved state in cells, after completion of culture, the cells are recovered by centrifugation, suspended in an aqueous buffer solution, and disrupted by a sonicator etc. Obtain an extract. From the supernatant obtained by centrifuging the cell-free extract, a conventional protein isolation and purification method, that is, a solvent extraction method, a salting out method with ammonium sulfate, a desalting method, a precipitation method with an organic solvent, Anion exchange chromatography method using resin such as diethylaminoethyl (DEAE) sepharose, cation exchange chromatography method using resin such as S-Sepharose FF (manufactured by Pharmacia), resin such as butyl sepharose or phenyl sepharose Obtain purified products using methods such as hydrophobic chromatography, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing, etc. alone or in combination. be able to.
By using a protein secretion expression system (CORYNEX (registered trademark): manufactured by Ajinomoto Co., Ltd.) using the above-mentioned Corynebacterium glutamicum, any complicated process such as crushing of cells to obtain a cell-free extract is not required, After centrifugation, the purified preparation can be obtained, for example, by purification using anion exchange chromatography (for example, HiTrap Q FF 5 mL FPLC column).

(The liver-type fatty acid binding protein preparation according to the seventh aspect)
The liver-type fatty acid-binding protein preparation according to the seventh aspect comprises an amino acid sequence having an identity of 90% or more with SEQ ID NO: 1 in the sequence listing, and an oxidation rate of methionine at 19th is 30% or more, or 113th methionine Contains a liver-type fatty acid binding protein having an oxidation rate of 70% or more.
As described later with reference to FIG. 13, when the oxidation rate of the 19th methionine is 30% or more, or the oxidation rate of the 113th methionine is 70% or more, a further increase in the oxidation rate is suppressed. The fluctuation of the binding ability due to the specifically binding substance can be suppressed.
In the liver-type fatty acid binding protein preparation according to the seventh aspect, even if it is a liver-type fatty acid binding protein preparation whose oxidation variation coefficient is set to 1.4 or less, the oxidation variation coefficient is set to 1.4 or less It may not be a liver-type fatty acid binding protein preparation, but is preferably a liver-type fatty acid binding protein preparation whose oxidation variation coefficient is set to 1.4 or less.
In the preparation of the liver-type fatty acid binding protein preparation according to the seventh aspect, when the oxidation rate of methionine 19 is set to 30% or more, from the viewpoint of being able to further suppress the fluctuation of the binding ability due to the specifically binding substance 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 acids other than methionine.
From the same point of view, at least one methionine selected from the group consisting of the 19th and the 74th is substituted with the above-mentioned nonpolar amino acids other than methionine when the oxidation rate of the 113th methionine is 70% or more. It does not have to be.
Further, the oxidation rate of methionine 19 is preferably 35% or more, more preferably 38% or more, from the viewpoint that variation in binding ability due to a specifically binding substance 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 still more preferably 80% or more.
As a method of measuring the oxidation rate, for example, MS spectrum of a peptide fragment containing Met19 in FIG. 6 (b) described later, MS spectrum of a peptide fragment containing Met 74 in FIG. 6 (c), Met 113 in FIG. It can be calculated from the comparison of the peak in the case of no oxidation treatment in the MS spectrum of the peptide fragment containing and the peak after oxidation treatment.
In addition, the liver-type fatty acid binding protein preparation is capable of suppressing the fluctuation of the binding ability due to the specifically binding substance regardless of the oxidation rate of methionine at position 19 and the oxidation rate of methionine at position 113. The oxidation rate of methionine at position 74 may be 60% or more.
In this case, the oxidation rate of the 74th methionine is preferably 65% or more, more preferably 70% or more, and the oxidation rate of the 74th methionine is 75% or more. The oxidation rate of methionine at position 74 is particularly preferably 80% or more, and the oxidation rate of methionine at position 74 is most preferably 85% or more.
The liver-type fatty acid binding protein preparation having the above-mentioned oxidation rate can be produced using an oxidizing agent such as AAPH or the like, or can be produced by air oxidation.

(The liver-type fatty acid binding protein preparation according to the eighth aspect)
In the liver-type fatty acid binding protein preparation according to the eighth aspect, the arachidonic acid, oleic acid, 8-isostataglandin F _2α and 2,3-dinor in an amount such that the oxidation variation coefficient is 1.4 or less. A liver comprising a liver-type fatty acid binding protein comprising at least one fatty acid selected from the group consisting of -8-isoprostaglandin F _2α and consisting of an amino acid sequence of 90% or more identity to SEQ ID NO: 1 in the sequence listing Type fatty acid binding protein preparation.
It is preferable that the amount by which the oxidation variation coefficient is 1.4 or less is an amount including 30 times or more the amount of the fatty acid with respect to the molar amount of the liver-type fatty acid binding protein.
In the liver-type fatty acid binding protein preparation according to the eighth aspect, even if it is a liver-type fatty acid binding protein preparation whose oxidation variation coefficient is set to 1.4 or less, the oxidation variation coefficient is set to 1.4 or less It may not be a liver-type fatty acid binding protein preparation, but is preferably a liver-type fatty acid binding protein preparation whose oxidation variation coefficient is set to 1.4 or less.
As described later with reference to FIG. 15, the present inventors changed the antibody binding ability of L-FABP protein depending on the type (FIG. 15 (b)) and concentration of fatty acid binding to L-FABP protein. I found it.
The liver-type fatty acid binding protein preparation according to the eighth aspect is selected from the group consisting of arachidonic acid, oleic acid, 8-isostataglandin F _2α and 2,3-dinor-8-isoprostaglandin F _2α The amount of at least one fatty acid is not more than 1.4 (preferably in the case of a plurality of fatty acids with respect to the molar amount of the liver-type fatty acid binding protein, at least 30 times the total content) In the case of expressing the L-FABP protein as a standard product by containing it in molar amounts, or in an assay system used as a standard, it is bound by an expression system, expression site or organ by different species of origin type fatty acids (e.g., arachidonic acid, oleic acid, 8 iso static prostaglandin _{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, it is specifically variations in binding ability due to the binding substance is suppressed, reference material for measuring, can function as a standard, such as control material.
From the viewpoint of further suppressing the fluctuation of the binding ability due to the specifically binding substance, arachidonic acid, oleic acid, 8-isostataglandin F _2α and 2,3-dinor relative to the molar amount of the hepatic fatty acid binding protein Preferably, the total content of at least one fatty acid selected from the group consisting of -8-isoprostaglandin F _2α is 50 times or more, preferably 75 times or more, in the case of multiple fatty acids. The amount is more preferably contained, further preferably 100 times or more, and particularly preferably 300 times or more.

More specifically, when the fatty acid is arachidonic acid, it is preferably contained 30 times or more, more preferably 50 times or more, more preferably 75 times or more, the molar amount of the liver-type fatty acid binding protein. It is further more preferable to contain the molar amount of 100, and it is particularly preferable to contain the molar amount of 100 times or more.
When the fatty acid is oleic acid, it is preferably contained 100 times or more, more preferably 300 times or more, and more preferably 1000 times or more the molar amount of the liver-type fatty acid binding protein. It is further preferable to include.
When the fatty acid is 8-isostastaglandin F _{2 α} , it is preferably contained 500 times or more, and more preferably 1000 times or more, the molar amount of the liver-type fatty acid binding protein.

In addition, when the preparation of liver-type fatty acid binding protein preparation according to the eighth aspect contains bovine serum albumin (BSA) as an adsorption inhibitor, arachidone is used relative to the molar amount of BSA from the viewpoint of adsorption of fatty acid to BSA. At least one type of 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 of multiple fatty acids, the total The content is preferably 0.02 to 10 times by mole, more preferably 0.2 to 5 times by mole.
When the preparation contains BSA and the fatty acid is arachidonic acid, the molar amount of arachidonic acid is preferably 1000 times or more, preferably 10000 times or more, the molar amount of the liver-type fatty acid binding protein. It is more preferable that the resin be contained in a molar amount of at least 100,000 times, and it is particularly preferable that the molar amount be at least 200,000 times.
When the fatty acid is oleic acid when the preparation contains BSA, it is preferable that oleic acid is contained in a molar amount of at least 100,000 times, preferably at least 200,000 times the molar amount of the liver-type fatty acid binding protein. More preferable.
When the preparation contains BSA and the fatty acid is 8-Isoprostaglandin F _2α , 8-Isoprostaglandin F _2α should contain a molar amount of at least 200,000 times the molar amount of the liver-type fatty acid binding protein Is preferred.
The liver-type fatty acid binding protein preparation according to the eighth aspect, which contains a specific amount of at least one type of fatty acid, is a liquid containing the liver-type fatty acid binding protein preparation, relative to the molar amount of the liver-type fatty acid binding protein. In the case of a plurality of fatty acids, the total content of at least one type of fatty acid is added in a molar amount of 30 times or more, or prepared by cell culture, protein isolation or purification conditions to achieve such content be able to. In addition, the content of the at least one type of fatty acid corresponds to the addition amount.
The upper limit value of the content of the fatty acid is not particularly limited, but when the following adsorption inhibitor is further contained, the fatty acid may be adsorbed to the adsorption inhibitor, so the molar amount of the liver-type fatty acid binding protein The fatty acid may be contained in a molar amount of 500,000 times, preferably not more than 300,000 times, more preferably not more than 200,000 times, and preferably not more than 10,000 times. Is more preferred.

The liver-type fatty acid binding protein preparation according to any one of the first, second, seventh and eighth aspects is characterized in that the measurement value of 37 ° C. in the measurement using a substance that specifically binds to the liver-type fatty acid binding protein The fluctuation range (for example, fluctuation due to oxidation) around 2 weeks is not particularly limited as long as the effects of the present invention are not impaired, but is preferably 15% or less, more preferably 10% or less. It is more preferably 5% or less, particularly preferably 1% or less.
The liver-type fatty acid binding protein preparation according to any one of the above first, second, seventh and eighth aspects optionally contains an adsorption inhibitor, an optional buffer solution, an optional surfactant and the like. It may be.
The adsorption inhibitor is not particularly limited as long as the effects of the present invention are not impaired, but includes BSA, casein, skim milk, polyethylene glycol and the like, with BSA being preferred.
The content of the adsorption inhibitor in the preparation of the liver-type fatty acid binding protein preparation according to any one of the first, second, seventh and eighth aspects is not particularly limited as long as the effects of the present invention are not impaired. It is preferable that it is 0.05-10 mass%.

The liver-type fatty acid binding protein preparation according to any one of the first, second, seventh and eighth aspects is a liver-type fatty acid binding protein to be offered for sale from the viewpoint of practical and commercial use. It is preferably a standard product. The liver-type fatty acid binding protein preparation to be offered for sale is not a sold protein, specifically a liver-type fatty acid binding protein preparation which has been left for a long time after being sold.
The liver-type fatty acid binding protein preparation according to any one of the first, second, seventh and eighth aspects measures a liver-type fatty acid binding protein in a sample by an immunological method utilizing an antigen-antibody reaction. For detecting or assaying L-FABP protein using binding specifically with an anti-L-FABP protein antibody that is useful as a standard substance for measurement of a kit or a substance for quality control and specifically binds to L-FABP protein It is preferable to use as a standard substance (standard product) of measurement of the
The measurement or detection of L-FABP protein for which the liver-type fatty acid binding protein preparation according to any one of the first, second, seventh and eighth aspects is used includes enzyme immunoassay (EIA, ELISA), fluorescent enzyme immunoassay (FLEIA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), immunochromatographic assay (ICA), latex Assays that employ agglutination (LA), fluorescent antibody (FA), radioimmunoassay (RIA), western blotting (WB), immunoblotting, etc. are mentioned, and the recognition site for the antigen (L-FABP protein) is Preferably, it is an assay employing a sandwich ELISA method using two different types of antibodies in combination.
Preferably, one of two types of antibodies having different recognition sites is used as a solid phase antibody bound to the surface of a well of a microplate, and the other is used as a labeled antibody for detection or quantification. There is no restriction | limiting in particular as a label | marker in the said labeled antibody, For example, enzyme label | markers, such as a peroxidase label | marker, a fluorescent label, an ultraviolet label, a radio label, etc. are mentioned.

  Examples of the antibody having a different recognition site for the antigen (L-FABP protein) include an antibody containing an antibody selected from the group consisting of anti-L-FABP antibody clone 1, clone 2, clone L and clone F. The combination containing FABP antibody clone L or the combination containing anti-L-FABP antibody clone 2 is preferable, the combination containing anti-L-FABP antibody clone L is more preferable, and the anti-L-FABP antibody clone L is It is further preferable to use as an immobilized antibody and any anti-L-FABP antibody as a labeled antibody, and to use an anti-L-FABP antibody clone L as an immobilized antibody and an anti-L-FABP antibody clone 2 as a labeled antibody. It is particularly preferred to use.

The L-FABP protein measurement kit used for such an assay comprises the liver-type fatty acid binding protein preparation according to any one of the first, second, seventh and eighth aspects as a preparation, and as a reagent It is preferable that the above-mentioned anti-L-FABP protein antibody is contained, and it is more preferable that a labeled anti-L-FABP protein antibody is further contained, and, if necessary, an adsorption inhibitor (BSA etc.), pretreatment solution (arbitrary buffer solution, arbitrary) And the like, a reaction buffer (any buffer and the like), a color forming substrate (3,3 ', 5,5'-tetramethylbenzidine, hydrogen peroxide water and the like), and the like.
As a L-FABP protein measurement kit, it is preferable to use a sandwich ELISA method in which two types of antibodies having different recognition sites for the antigen are combined and used, and any anti-L-FABP antibody or labeled antibody may be used as a solid phase. More preferably, it is a kit using L-FABP antibody clone 2.
As a specific embodiment of the L-FABP protein measurement kit using a sandwich ELISA method, for example, a kit including the following (1) to (9) can be mentioned.
(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 (derived from clone 2 producing cell line)
(5) Enzyme substrate solution (6) Detergent (optional buffer, surfactant, etc.)
(7) Reaction stop solution (1 N sulfuric acid etc.)
(8) Standard buffer (optional 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, but any liquid may be used. A solution prepared 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 is, for example, 10 to 10000 ng / mL, preferably 50 to 5000 ng / mL, 100 to 500 1000 ng / mL is more preferable, 200 to 800 ng / mL is further preferable, and 300 to 600 ng / mL is particularly preferable.
The liver-type fatty acid binding protein preparation according to any one of the above first, second, seventh and eighth aspects is used except that the conventional recombinant human L-FABP is used as a liver-type fatty acid binding protein preparation. As a commercially available product of a kit similar to the L-FABP protein measurement kit using the sandwich ELISA method described above, "Lenapro L-FABP Test TMB" (manufactured by Cimic Holdings, Inc.) can be mentioned.

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

<Method for evaluating liver-type fatty acid binding protein preparation using oxidation variation coefficient as index>
The method for evaluating a liver-type fatty acid binding protein preparation according to the ninth aspect is the oxidation represented by the ratio of the measurement value after the oxidation treatment to the measurement value using the liver-type fatty acid binding protein preparation before the oxidation treatment Using the coefficient of variation as an index, evaluate the difficulty of oxidation variation of the above-mentioned standard.
In the method for evaluating a liver-type fatty acid binding protein preparation according to the ninth aspect, the oxidation variation coefficient is preferably 1.4 or less, and is 1.3 or less, from the viewpoint of suppression of the oxidation variation range. More preferable.
The lower limit value of the oxidation variation coefficient is not particularly limited as long as the effects of the present invention are not impaired, but, for example, 0.8 or more can be mentioned, preferably 0.9 or more, and 1.0 or more. More preferable.

<Method for suppressing the fluctuation of measured value in measurement using liver-type fatty acid binding protein preparation>
According to a tenth aspect of the present invention, there is provided a method of suppressing the fluctuation range of the measurement value attributed to a hepatic fatty acid binding protein in the measurement using a hepatic fatty acid binding protein preparation according to the tenth aspect,
(1) At least one methionine in the 19th, 74th and 113th positions of the liver-type fatty acid binding protein consisting of an amino acid sequence of 90% or more identity to SEQ ID NO: 1 in the sequence listing is substituted with nonpolar amino acids other than methionine And replacing at least the 19th methionine with a nonpolar amino acid other than methionine,
(2) The oxidation rate of methionine of the 19th methionine of the liver-type fatty acid binding protein consisting of an amino acid sequence of 90% or more identity to SEQ ID NO: 1 in the sequence listing is 30% or more, or the oxidation rate of methionine of the 113th is 70% or more And (3) at least one fatty acid selected from the group consisting of arachidonic acid, oleic acid, 8-isostataglandin F _2α and 2,3-dinor-8-isoprostaglandin F _2α It contains at least one selected from the group consisting of including the above-mentioned liver-type fatty acid binding protein preparation.
In the method according to the tenth aspect, the oxidation rate of methionine at position 19 is preferably 35% or more, more preferably 38% or more, still more preferably 40% or more, and 45% or more Is particularly preferred.
The oxidation rate of the 113th methionine is preferably 73% or more, more preferably 75% or more, and still more preferably 80% or more.
In the method according to the tenth aspect, the content of the at least one type of fatty acid to be contained in the preparation of liver-type fatty acid binding protein is not particularly limited as long as the effects of the present invention are not impaired. The molar amount is preferably 30 times or more, more preferably 50 times or more, still more preferably 75 times or more, and further preferably 100 times or more the molar amount of the protein. It is particularly preferable that the molar amount is 300 times or more.
The upper limit of the content of the fatty acid is not particularly limited, but when the adsorption inhibitor is further contained, the fatty acid may be adsorbed to the adsorption inhibitor, so the molar amount of the liver-type fatty acid binding protein The fatty acid can be contained in a molar amount of 500,000 times, preferably not more than 200,000 times, more preferably not more than 100,000 times, and preferably not more than 10,000 times. Is more preferable, and a molar amount of 1000 times or less is particularly preferable.
According to the method of the tenth aspect, the fluctuation range of the measurement value in the measurement using the substance that specifically binds to the hepatic fatty acid binding protein can be suppressed, and preferably the fluctuation at around 37 ° C. for two weeks The width may be 15% or less, more preferably 10% or less, more preferably 5% or less, and particularly preferably 1% or less. it can.

<Method of Preparing a Calibration Curve and Method of Quantifying Liver-Type Fatty Acid-Binding Protein>
The method for preparing a calibration curve of a liver-type fatty acid binding protein according to the eleventh aspect uses the preparation of a liver-type fatty acid binding protein according to any one of the first, second, seventh and eighth aspects.
Specifically, a calibration curve is created based on the relationship between the intensity of the label to be measured (for example, enzyme labeling intensity, fluorescence intensity, ultraviolet intensity, radiation intensity, etc.) and the amount of the standard (for example, concentration). can do.
The method for quantifying a liver-type fatty acid binding protein in a sample according to the twelfth aspect uses a calibration curve prepared by the method according to the eleventh aspect.
Specifically, the labeling intensity of the liver-type fatty acid binding protein in a sample labeled with a labeled antibody or the like is measured under the same conditions as the preparation of the above calibration curve, and based on the above calibration curve (for example, comparison) Hepatic fatty acid binding protein in a sample can be detected or quantified.

  Hereinafter, the present invention will be described in more detail by way of examples, but the scope of the present invention is not limited to these examples.

Reference example (Structure change of L-FABP protein by oxidation)
L-FABP protein was treated with AAPH. The results are shown in FIGS. 2 (a) and (b).
Fig.2 (a) is a figure which shows the molecular weight measurement result by LC-ESI-MS of oxidized L-FABP and nonoxidized L-FABP, FIG.2 (b) is oxidized L-FABP and nonoxidized L -Figure which shows the fluorescence spectrum of FABP.
As apparent from FIG. 2A, in the oxidized L-FABP, an increase in molecular weight corresponding to approximately 2 to 3 oxygen molecules is observed from the theoretical molecular weight.
As shown in FIG. 2 (b), in oxidized L-FABP, a fluorescence peak appears around 350 nm (arrow in the figure), and the environment with high polarity around the aromatic amino acid present in L-FABP protein It is considered that the oxidized L-FABP and the nonoxidized L-FABP have undergone a structural change.

(Change of ELISA measurement value by oxidation)
AAPH was added to the L-FABP protein solution to a predetermined concentration, reacted at room temperature for 1 hour, and ELISA measurement was performed. The results are shown in FIG.
It can be seen from FIG. 3 that the AAPH treatment of the L-FABP protein causes a change in the ELISA measurement value (ratio (%) when taking no treatment as 100).
Also, surprisingly, as shown in FIG. 3, the ELISA measurement value is increased by reacting for 1 hour at room temperature also in the AAPH additive-free sample (with H ₂ O added), and the effect of air oxidation is It is considered to have occurred.

(Oxidation of human L-FABP protein)
Various concentrations (40 mM, 200 mM) of AAPH were added to human L-FABP protein and reacted at 37 ° C. for 1.5 hours. The results are shown in FIG.
FIG. 4 shows MS spectra after adding various concentrations (40 mM, 200 mM) of AAPH to human L-FABP protein and reacting at 37 ° C. for 1.5 hours.
As shown in FIG. 4, an increase in molecular weight corresponding to 1 to 3 oxygen molecules was observed in L-FABP by AAPH treatment.

(Oxidation of methionine contained in human L-FABP protein)
FIG. 5 (a) shows the amino acid sequence of human L-FABP protein shown in SEQ ID NO: 1, and FIG. 5 (b) shows the estimation of various peptide fragments which can be produced by tryptic digestion of 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. 6 (a) shows Met 1 containing peptide fragment No. 1 obtained by trypsin digestion of human L-FABP protein after reaction with the above-mentioned various concentrations (40 mM, 200 mM) of AAPH. FIG. 6 (b) is a diagram showing a Met19-containing peptide fragment No. 1 obtained by trypsin digestion of human L-FABP protein after reaction with the above-mentioned various concentrations of AAPH. FIG. 6 (c) is a diagram showing a Met74-containing peptide fragment No. 5 containing Met74 obtained by trypsin digestion of human L-FABP protein after reaction with the above-mentioned various concentrations of AAPH. 9 and peptide fragment no. FIG. 6 (d) is a diagram showing a peptide fragment No. 10 containing Met113 obtained by trypsin digestion of human L-FABP protein after reaction with the above-mentioned various concentrations of AAPH. 14 shows MS spectra of 14 and peptide fragment 15. FIG.
From the results shown in FIGS. 6A to 6D, 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.

Example 1
(Method for producing mutant L-FABP protein)
A mutated protein in which Met19 is mutated to a leucine residue (hereinafter also referred to as L-FABP M19L),
A mutated protein in which Met74 is mutated to a leucine residue (hereinafter also referred to as L-FABP M74L),
A mutated protein in which Met113 is mutated to a leucine residue (hereinafter also referred to as L-FABP M113L),
Mutant proteins in which Met19 and Met74 have been mutated to leucine residues, respectively (hereinafter also referred to as L-FABP M19L / M74L),
Mutant proteins in which Met19 and Met113 are mutated to leucine residues respectively (hereinafter also referred to as L-FABP M19L / M113L),
Mutant proteins in which Met74 and Met113 have been mutated to leucine residues, respectively (hereinafter also referred to as L-FABP M74L / M113L),
A mutant protein (hereinafter also referred to as L-FABP M19L / M74L / M113L) in which three methionine residues (Met19, Met74, Met113) were mutated to leucine residues, respectively, was produced.
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 respectively express their expression using a protein secretion expression system (CORYNEX (registered trademark): made by Ajinomoto Co., Ltd.) 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 following sequence listing.
The gene sequence of L-FABP M19L / M74L / M113L used for protein expression is shown in SEQ ID NO: 3 in the following sequence listing.

L-FABP M19L / M74L / M113L etc. which were expressed by the method mentioned above first use centrifugal filter unit Amicon (registered trademark) Ultra-15, 3 kDa (manufactured by Millipore) and buffer A (10 mM Tris-HCl (pH 8) .5) Buffer exchange with 1 mM DTT).
Next, purification was performed using a HiTrap Q FF column, 5 mL (manufactured by GE Healthcare). L-FABP M19L / M74L / M113L adsorbed on HiTrapQ FF column was washed with buffer A, then linear concentration gradient method with buffer A and buffer B (10 mM Tris-HCl (pH 8.5), 1 mM DTT, 2 M NaCl) The fractions containing the peak at which buffer B was 3.1% were collected. 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. The purification results for L-FABP M19L / M74L / M113L are shown in FIG.

  L-FABP M19L / M74L / M113L etc. which were obtained by the above-mentioned operation were stored at -80 ° C.

L-FABP protein solution (L-FABP M19L, L-FABP M74L, L-FABP M113L, L-FABP M19L / M74L, so that AAPH reaches a predetermined final concentration (0 mM, 0.5 mM, 1 mM, 2 mM, 4 mM) Add to L-FABP M19L / M113L, L-FABP M74L / M113L, and L-FABP M19L / M74L / M113L), react at room temperature for 1 hour, perform ELISA measurement, and develop colored antibody (OD450nm) ) Was compared to the untreated sample. The comparison results are shown in FIG.
As is clear from the results shown in FIG. 8, it can be seen that the stability against oxidation is improved in L-FABP M19L and L-FABP M74L, and the oxidation rates of the 19th and 113th methionines are dominant. confirmed. Furthermore, it is understood that L-FABP M19L / M74L / M113L is most excellent in oxidation stability.
Next, L-FABP M19L / M74L / M113L etc. whose oxidation stability has been confirmed are dissolved in the above-mentioned protein storage buffer for long-term storage of samples, and stored at -80 ° C. And 8 were used.

Example 2
(Stability to oxidation)
In the presence of BSA, AAPH was added to L-FABP M19L / M74L / M113L to a final concentration of 0-5 mM, and reacted at room temperature for 1 hour. In addition, in consideration of the influence of air oxidation, only H ₂ O was added to the protein solution, and the untreated sample in which the measurement was performed 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 Cimic Holdings Co., Ltd.) to compare the color development of the labeled antibody (OD 450 nm) with the untreated samples. The method of using the above-mentioned diagnostic kit was carried out according to the measurement method according to the attached document usually attached. The results are shown in FIG.

As is clear from the results of the ELISA measurement shown in FIG. 9, the L-FABP WT was confirmed to have an increase 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 102-107%, and CV was 2.4%.
From the above results, it was revealed that the antibody binding ability of L-FABP M19L / M74L / M113L to AAPH is stable, 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 the oxidation reaction. In addition, the antibody binding ability of L-FABP WT is increased only by placing it at room temperature for 1 hour, but L-FABP M19L / M74L / M113L does not increase, so it can be said that it is stabilized against air oxidation.

(Stability at room temperature)
L-FABP M19L / M74L / M113L was stored at room temperature (25 ° C.) in the presence of BSA, and ELISA measurement was performed according to a standard method using “Lenapro L-FABP test TMB” up to 4 weeks after every week . The measurement was also performed on a sample stored at -80 ° C, and was used as a comparison target. About the color development (OD450nm) of the labeled antibody of a room temperature storage sample, the ratio (%) which made the color development (OD450nm) of the labeling antibody of a -80 degreeC storage sample into 100 was compared. The results are shown in FIG.

As is clear from the results of the ELISA measurement shown in FIG. 10, L-FABP WT was confirmed to have an increase of 110 to 128%, and CV was 10.5%.
On the other hand, L-FABP M19L / M74L / M113L was observed only up to 101 to 111%, and CV was 4.7%.
From the above results, it was revealed that the antibody binding ability of L-FABP M19L / M74L / M113L is stable against long-term storage at room temperature, and the fluctuation of the ELISA measurement value is small. That is, it can be said that L-FABP M19L / M74L / M113L was stabilized by long-term storage at room temperature.

(Stability at 37 ° C)
L-FABP M19L / M74L / M113L was stored at 37 ° C. in the presence of BSA, and ELISA measurement was performed according to a standard method using “Lenapro L-FABP test TMB” every week until 4 weeks. The measurement was also performed on a sample stored at -80 ° C, and was used as a comparison target. About the color development (OD450nm) of the labeled antibody of a room temperature storage sample, the ratio (%) which made the color development (OD450nm) of the labeling antibody of a -80 degreeC storage sample into 100 was compared. The results are shown in FIG.

As is clear from the results of the ELISA measurement shown in FIG. 11, L-FABP WT was confirmed to have an increase of 117 to 143%, and CV was 14.5%.
On the other hand, L-FABP M19L / M74L / M113L was observed only up to 104 to 113%, and CV was 4.8%.
From the above results, it was revealed that the antibody binding ability of L-FABP M19L / M74L / M113L is stable against long-term storage at 37 ° C., and the fluctuation of the ELISA measurement value is small. That is, it can be said that L-FABP M19L / M74L / M113L was stabilized also for long-term storage at 37 ° C.

Example 3
<Oxidation stability in ELISA measurement using a labeled antibody having a recognition site different from that of clone 2>
AAPH was added to L-FABP protein solution to a predetermined final concentration (0 to 4 mM), reacted at room temperature for 1 hour, and ELISA measurement was performed. The color development (OD 450 nm) was compared to an untreated sample using clone 1, clone 2 and clone F as labeled antibodies. The results are shown in FIG.
As is clear from the results shown in FIG. 12, in the L-FABP measurement method using the labeled antibody with clone 1 and clone F, the antibody binding ability of L-FABP WT is changed by oxidation as in clone 2. It is found that L-FABP M19L / M74L / M113L suppresses the change in antibody binding ability due to oxidation.
Therefore, L-FABP protein stabilized also in the ELISA measurement system by anti-L-FABP antibody clone 1 and anti-L-FABP antibody clone F which differ in an antigen recognition site is effective.

ELISA measurement is carried out in the same manner as in Example 3 by the sandwich ELISA method using the above-mentioned anti-L-FABP antibody clone 2 as a labeled antibody, and the 19th, 74th and 113th of various L-FABP proteins different in the progress of oxidation The oxidation rate of methionine was measured. The results are shown in FIG.
In FIG. 13, * indicates that the oxidation is saturated and stabilized based on data of ELISA measurement value of WT L-FABP using the clone 2 shown in Example 3 and FIG. 12 (AAPH added concentration data). It is the area which made the range of the mean value ± 2SD (standard deviation) as "the saturation area of the rise in ELISA reactivity by oxidation" as the
As can be seen from FIG. 13, the minimum oxidation rate in the region where oxidation is saturated and stabilized is about 38% for the 19th methionine and about 70% for the 74th methionine, and 113th Was about 73% for methionine.
From the above results, it can be said that having an oxidation rate of 30% or more for the 19th methionine contributes to the reduction of the fluctuation range of the ELISA measurement value.
In addition, it can be said that the 113th methionine having an oxidation rate of 70% or more also contributes to the reduction of the fluctuation range of the ELISA measurement value.
Further, from the results shown in FIG. 13, it is possible to predict that the increase in the ELISA measurement value is saturated in the “saturation region of the increase in ELISA reactivity due to oxidation” also in the 74th methionine.

Example 4
<Stability of L-FABP preparation subjected to oxidation treatment at 37 ° C>
L-FABP oxidized with 40 mM AAPH was stored at 37 ° C., and ELISA measurement was performed according to a standard method using “Lenapro L-FABP test TMB” every two weeks up to two weeks later. The measurement was also performed on a sample stored at 4 ° C., and was used as a comparison target. The percentage (%) of the color development (OD450 nm) of the labeled antibody of the 4 ° C. storage sample relative to the color development (OD 450 nm) of the labeled antibody of the 37 ° C. storage sample was compared. The results are shown in FIG.

As apparent from the results of the ELISA measurement shown in FIG. 14, L-FABP subjected to oxidation treatment was observed only up to 94 to 102%. On the other hand, L-FABP which has not been oxidized was found to increase by 114 to 130%.
From the above results, it was revealed that the antibody binding ability of oxidized L-FABP was stable for long-term storage at 37 ° C., and the fluctuation of the ELISA measurement value was small. That is, it can be said that L-FABP subjected to oxidation treatment was stabilized also for long-term storage at 37 ° C.

Example 5
<Stability by fatty acid addition>
FIG. 15 (a) is a figure which shows the change (The ratio (%) which made the fatty acid addition of the quantity of 0 times the amount 100) by the fatty acid addition process of L-FABP protein.
It became clear that the antibody binding ability of L-FABP protein was changed depending on the type of fatty acid to be bound (FIG. 15 (b)) and the concentration.
FIG. 15 (a) shows the antibody binding ability to L-FABP protein for 8-isostataglandin F _2α , 2,3-dinor-8-isostataglandin F _2α and enta-prostaglandin E ₂ Indicates a change. Arachidonic acid and oleic acid will be described later.

  Next, the final molar amount of each type of fatty acid is added to 200,000 times the molar amount of wild-type L-FABP protein (L-FABP WT) or L-FABP M19L / M74L / M113L, and it is 1.5 at room temperature It was made to react for time. Also, in consideration of the influence of air oxidation, only DMSO (final concentration 5%) was added to the protein solution, and an untreated sample in which the measurement was performed immediately after the addition was used as a comparison target sample.

  These reaction solutions and untreated samples were subjected to ELISA measurement in the presence of 1% by mass BSA using “Lenapro L-FABP Test TMB” to compare the color development of the labeled antibody (OD450 nm) with the untreated samples. The method of using the above-mentioned diagnostic kit was carried out according to the measurement method according to the attached document usually attached. The results are shown in FIGS. 16 (a) to 16 (c).

Fig.16 (a) is a figure which shows the change of the ELISA measurement value by the arachidonic acid addition of various concentration, FIG.16 (b) is a figure which shows the change of the ELISA measurement value by the oleic acid addition of various concentration. (c) is a graph showing changes in ELISA measurements by 8 iso static prostaglandin _{F 2.alpha} addition of various concentrations.
As apparent from the results of the ELISA measurement shown in FIG. 16 (a) to (c), L-FABP WT shows a 104-220% increase in measured value for arachidonic acid (CV 34.0%) and for oleic acid There was a 100% to 124% increase in measured value (CV 10.4%) and a variation in measured value of 95 to 118% (CV 6.8%) for 8-Isoprostaglandin F ₂ α.
On the other hand, L-FABP M19L / M74L / M113L has a variation of measured value of 88 to 103% (CV6.1%) for arachidonic acid, and a variation of measured value of 92 to 107% for oleic acid, (CV5.1% For 8-Isoprostaglandin F _2α , an increase of 96 to 101% (CV 2.3%) was observed.
From the results shown in FIG. 16, as L-FABP WT is added with fatty acid, the change in 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 standard Thus, it can be seen that the fluctuation range of the ELISA measurement value can be suppressed.
In particular, from the results shown in FIG. 16 (a), it can be seen that, when arachidonic acid is contained, the change in the ELISA measurement value approaches saturation at a relatively small molar amount.
In addition, it is assumed 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 was stable with respect to fatty acid addition, and the fluctuation | variation of ELISA measured value was small. That is, it can be said that L-FABP M19L / M74L / M113L was stabilized against fatty acid addition.

Example 6
<Suppression of fluctuation of ELISA measurement value by binding fatty acid (arachidonic acid or oleic acid) to L-FABP>
FIG. 17 and FIG. 18 are diagrams showing the addition amount of fatty acid (arachidonic acid or oleic acid) and the change in ELISA measurement value (ratio (%) where the amount of fatty acid addition is 0 molar times 100).
0 to 100 molar volumes of fatty acid (arachidonic acid or oleic acid) relative to L-FABP are added in the absence of BSA, reacted at room temperature for 60 minutes, and then dialyzed against saline overnight did. The next day, the dialysis outer solution was replaced and dialysis was performed again overnight to remove free fatty acid not bound to L-FABP. Changes in ELISA measurement values of the obtained sample (the ratio (%) where the amount of fatty acid added is 0 molar times 100) are shown in FIGS.
As apparent from FIG. 17, the change in the ELISA measurement value by the addition of arachidonic acid became constant when 10 or more molar amount of fatty acid was added to L-FABP, and 30 or more molar amount of arachidonic acid was added. It was confirmed that it became more constant.
Further, as apparent from FIG. 18, the change in the ELISA measurement value due to the addition of oleic acid becomes constant when 100 molar times or more amount of fatty acid is added to L-FABP, and 300 molar times or more oleic acid It was confirmed that it becomes more constant when added.

Example 7
<Stability of L-FABP preparation bound with arachidonic acid or oleic acid at 37 ° C.>
Using a sample prepared by diluting L-FABP containing 50 times molar amount of arachidonic acid with respect to the molar amount of L-FABP in a manner similar to Example 6 and then binding to a BSA-containing protein storage buffer The sample was stored in the same manner as in Example 5, and changes in ELISA measurements at 37.degree. C. for two weeks were confirmed. The results are shown in FIG.
Similarly, the L-FABP containing and conjugated with a molar amount of oleic acid of 1000 times the molar amount of L-FABP was diluted in a BSA-containing protein storage buffer and prepared by the same method. Changes in ELISA measurements at 37 ° C. for 2 weeks were confirmed. The results are shown in FIG.

As apparent from the results of the ELISA measurement shown in FIG. 19, L-FABP bound with arachidonic acid showed only a fluctuation of 99.9 to 100.3%. On the other hand, the L-FABP not bound with arachidonic acid was found to increase by 118 to 132%.
From the above results, it was revealed that the antibody binding ability of L-FABP bound with arachidonic acid was stable against long-term storage at 37 ° C., and the variation of the measured value of ELISA was small.
Similarly, as apparent from the results of the ELISA measurement shown in FIG. 20, L-FABP bound to oleic acid showed only a variation of 100 to 110%. On the other hand, L-FABP which did not bind oleic acid showed a 120-130% increase.
From the above results, it was revealed that the antibody binding ability of L-FABP conjugated with oleic acid is stable against long-term storage at 37 ° C., and the fluctuation of the ELISA measurement value is small.

Example 8
<Evaluation of L-FABP standard using oxidation variation coefficient as index>

L-FABP M19 L / M 74 L / M 113 L in which the fluctuation of the ELISA measurement value is shown to be small in Example 3 and FIG. 12 L-FABP WT treated with 0.5 mM AAPH, L- treated with 4 mM AAPH The FABP WT and L-FABP WT were subjected to 1 hour oxidation treatment with 10 mM AAPH at 25 ° C., and the oxidation variation coefficient was calculated from the OD ratio of the ELISA measurement value with or without the above oxidation treatment. Moreover, it calculated also about L-FABP WT as a control. The results are shown in FIG.
As apparent from the results shown in FIG. 21, the oxidation variation coefficient of L-FABP WT as a control was about 1.8.
On the other hand, L-FABP M19L / M74L / M113L shown in Example 3 and FIG. 12 shows a small fluctuation of the ELISA measurement value L-FABP WT treated with 0.5 mM AAPH, L treated with 4 mM AAPH -All of FABP WT had an oxidation variation coefficient of 1.3 or less.
Therefore, L-FABP WT treated with L-FABP M 19 L / M 74 L / M 113 L, 0.5 mM AAPH, mean value + 2 SD (standard deviation) of oxidation variation coefficient of L-FABP WT treated with 4 mM AAPH. When it is 4 (1.2 + 0.2) or less, it can be said that the fluctuation of the measured value is suppressed against oxidation and stable.

Next, each of various L-FABP proteins different in the progress of oxidation used in Example 3 and FIG. 13 is subjected to oxidation treatment with 10 mM AAPH for 1 hour at 25 ° C., and the OD of the ELISA measurement value with or without the oxidation treatment 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 is represented by the remaining unoxidized methionine content rate (100% -methionine oxidation rate) and the oxidation variation coefficient Calculated as the correlation of The results are shown in FIG.
As is clear from the results shown in FIG. 22, the oxidation variation coefficient converges at the remaining unoxidized methionine content of less than 70% (that is, the oxidation ratio of 30% or more) for the 19th methionine. It can be seen that the oxidation fluctuation is suppressed.
In particular, when the residual unoxidized methionine content is less than 62% (that is, the oxidation rate is 38% or more), which is a case where the oxidation variation coefficient is 1.4 or less, it can be said that suppression of oxidation variation is particularly excellent.

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

In addition, (1) L-FABP WT, (2) oleic acid-containing L-FABP in Examples 6 and 7, (3) arachidonic acid-containing L-FABP, (4) L-FABP treated with 0.5 mM AAPH Oxidation variation coefficients for WT, (5) L-FABP WT and (6) L-FABP M19L / M74L / M113L, (7) L-FABP M19L, and (8) L-FABP M19L / M113L treated with 4 mM AAPH Was calculated. The calculation results are shown in FIG.
As is clear from the results shown in FIG. 23, the L-FABP WT has an oxidation variation coefficient much larger than 1.4 (about 1.8) and does not satisfy the stability against oxidation as a L-FABP standard. I understand.
On the other hand, (2) L-FABP containing oleic acid, (3) L-FABP containing arachidonic acid, (4) L-FABP WT treated with 0.5 mM AAPH, (5) L-FABP treated with 4 mM AAPH WT and (6) L-FABP M19L / M74L / M113L, (7) L-FABP M19L, and (8) L-FABP M19L / M113L all have an oxidation variation coefficient of 1.4 or less, and stability against oxidation It is understood that it is excellent.

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 (15)

  The oxidation variation coefficient represented by the ratio of the measured value obtained by the oxidation treatment to the measured value using a liver-type fatty acid binding protein preparation not oxidized at 25 ° C. for 1 hour with 10 mM oxidizing agent is 1.4 or less Liver-type fatty acid binding protein standard set in   An amino acid sequence having an identity of 90% or more with SEQ ID NO: 1 of the sequence listing, which is used for the preparation of liver-type fatty acid binding protein preparation according to claim 1, and one or more methionine of the 19th, the 74th and the 113th A liver-type fatty acid binding protein substituted with a nonpolar amino acid other than methionine, wherein at least the 19th methionine is substituted with a nonpolar amino acid other than methionine.   A DNA encoding the protein according to claim 2.   A cell transformed with the DNA of claim 3.   A method for producing a protein according to claim 2, comprising the steps of culturing the cell according to claim 4 and recovering the protein according to claim 2.   It consists of an amino acid sequence of 90% or more identity to 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 The liver-type fatty acid binding protein preparation according to claim 1, which comprises a liver-type fatty acid binding protein having an oxidation rate of 70% or more of methionine. Arachidonic acid in an amount of the oxidizing variation coefficient is 1.4 or less, oleic acid, at least is selected from the group consisting of 8-iso static Prostaglandin F _2.alpha and 2,3-dinor-8-iso static prostaglandin F _2.alpha The liver-type fatty acid binding protein preparation according to claim 1, comprising a liver-type fatty acid-binding protein comprising one type of fatty acid and consisting of an amino acid sequence having an identity of 90% or more with SEQ ID NO: 1 in the sequence listing.   The liver type according to claim 7, wherein the amount that the oxidation variation coefficient becomes 1.4 or less is an amount containing the amount of the fatty acid in a molar amount of 30 times or more to the molar amount of the hepatic fatty acid binding protein. Fatty acid binding protein preparation.   The liver-type fatty acid binding according to any one of claims 1 and 6 to 8, wherein a fluctuation range of around 37 ° C for 2 weeks in measurement value in measurement using a substance that specifically binds to liver-type fatty acid binding protein is 10% or less. Protein preparation.   The liver-type fatty acid binding protein preparation according to any one of claims 1 and 6 to 9 for sale.   The liver-type fatty acid binding protein preparation according to any one of claims 1 and 6 to 10, which is used as a measurement standard substance or a quality control substance.   A method of evaluating a liver-type fatty acid binding protein preparation by using an oxidation variation coefficient represented by a ratio of a measurement value obtained by performing the oxidation processing to a measurement value using a liver-type fatty acid binding protein preparation which is not oxidized. A method for suppressing the fluctuation range of measurement values in measurement using a liver-type fatty acid binding protein preparation,
(1) At least one methionine in the 19th, 74th and 113th positions of the liver-type fatty acid binding protein consisting of an amino acid sequence of 90% or more identity to SEQ ID NO: 1 in the sequence listing is substituted with nonpolar amino acids other than methionine And replacing at least the 19th methionine with a nonpolar amino acid other than methionine,
(2) The oxidation rate of methionine at position 19 of the liver-type fatty acid binding protein consisting of an amino acid sequence of 90% or more identity to SEQ ID NO: 1 in the sequence listing is 30% or more, or the oxidation rate of methionine at position 19 Making the oxidation rate of methionine of 30% or more and 70% or more of the 113th methionine, and (3) arachidonic acid, oleic acid, 8-isostataglandin F _2α and 2,3-dinor-8-isoprostagran A method comprising at least one selected from the group consisting of including in the liver-type fatty acid binding protein preparation at least one fatty acid selected from the group consisting of gin F _2α .   A method of preparing a calibration curve of liver-type fatty acid binding protein using the liver-type fatty acid binding protein preparation according to any one of claims 1 and 6 to 11.   A method of quantifying a liver-type fatty acid binding protein in a sample using a calibration curve prepared by the method according to claim 14.