JP6059388B1 - Liver-type fatty acid binding protein, DNA encoding the protein, cells transformed with the DNA, a method for producing the protein, a preparation of the protein, a method for preparing a calibration curve for the protein, and quantifying the protein Method - Google Patents
Liver-type fatty acid binding protein, DNA encoding the protein, cells transformed with the DNA, a method for producing the protein, a preparation of the protein, a method for preparing a calibration curve for the protein, and quantifying the protein Method Download PDFInfo
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- JP6059388B1 JP6059388B1 JP2016120073A JP2016120073A JP6059388B1 JP 6059388 B1 JP6059388 B1 JP 6059388B1 JP 2016120073 A JP2016120073 A JP 2016120073A JP 2016120073 A JP2016120073 A JP 2016120073A JP 6059388 B1 JP6059388 B1 JP 6059388B1
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Abstract
【課題】幅広い保存条件において、特異的に結合する抗体による結合能の安定性に優れる肝型脂肪酸結合蛋白質、該蛋白質を用いた標品、該蛋白質をコードするＤＮＡ、該ＤＮＡで形質転換された細胞、該蛋白質の検量線を作成する方法、及び該蛋白質を定量する方法の提供を提供すること。【解決手段】配列表の配列番号１と相同性９０％以上のアミノ酸配列からなり、１９番目、７４番目、１１３番目の１つ以上のメチオニンが、メチオニン以外の非極性アミノ酸に置換された肝型脂肪酸結合蛋白質。【選択図】図１A liver-type fatty acid binding protein having excellent stability of binding ability by an antibody that specifically binds under a wide range of storage conditions, a preparation using the protein, a DNA encoding the protein, and transformed with the DNA To provide a cell, a method for preparing a calibration curve for the protein, and a method for quantifying the protein. A liver type comprising an amino acid sequence having a homology of 90% or more with SEQ ID NO: 1 in the sequence listing, wherein one or more methionines at the 19th, 74th, and 113th positions are substituted with nonpolar amino acids other than methionine. Fatty acid binding protein. [Selection] Figure 1
Description
本発明は、特異的に結合する抗体による結合能が安定化された肝型脂肪酸結合蛋白質、該蛋白質をコードするＤＮＡ、該ＤＮＡで形質転換された細胞、該蛋白質の製造方法、該蛋白質を用いた標品、該蛋白質の検量線を作成する方法、及び該蛋白質を定量する方法に関するものである。 The present invention relates to a liver-type fatty acid binding protein in which the binding ability of an antibody that specifically binds is stabilized, a DNA encoding the protein, a cell transformed with the DNA, a method for producing the protein, and the use of the protein The present invention relates to a standard preparation, a method for preparing a calibration curve for the protein, and a method for quantifying the protein.
脂肪酸結合蛋白質（Ｆａｔｔｙ Ａｃｉｄ−Ｂｉｎｄｉｎｇ Ｐｒｏｔｅｉｎ；以下ＦＡＢＰともいう。）は細胞内脂質結合蛋白質ファミリーに属する分子量約１４ｋＤａの蛋白質で、脂肪酸をはじめとする疎水性リガンドと可逆的に結合し細胞内輸送を担うことが知られている（例えば、非特許文献１）。その中でも肝型脂肪酸結合蛋白質（Ｌ−ｔｙｐｅ ｆａｔｔｙ ａｃｉｄ−ｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ；以下、単に「Ｌ−ＦＡＢＰ蛋白質」ともいう。）は肝臓や腎臓の近位尿細管細胞の細胞質に局在しており、尿細管障害による虚血・酸化ストレスに応答して尿中への排泄量が増加する（例えば、非特許文献２）。そのため尿中の腎臓組織由来Ｌ−ＦＡＢＰ蛋白質を検出することで腎疾患の検査が可能である（例えば、特許文献１）。
また、図１５に示したように、Ｌ−ＦＡＢＰ蛋白質は、逆平行βシートが２枚直行したβバレル構造に２本のαへリックスが蓋をするような形で安定化され、２分子の遊離脂肪酸（例えば、オレイン酸）と結合することが知られている（ＰＤＢ ＩＤ：２ＬＫＫ）（非特許文献３）。
そして、Ｌ−ＦＡＢＰ蛋白質は遊離脂肪酸をミトコンドリアやペルオキシソームへ輸送し、β酸化を促進する機構を有している（非特許文献４）。
脂肪酸リガンドの結合親和性としては、脂肪酸の炭素鎖が伸長し、二重結合が増えるほど高くなる傾向が見られ（非特許文献５、６）、Ｌ−ＦＡＢＰ蛋白質は特に過酸化物に対して結合親和性が高いことなどが報告されている（非特許文献７）。
また、Ｌ−ＦＡＢＰ蛋白質の抗酸化機構を研究した報告では、ＡＡＰＨによってラットＬ−ＦＡＢＰ蛋白質のメチオニン残基が酸化されていたことが示されている（非特許文献８）。
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).
Further, as shown in FIG. 15, 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 orthogonally crossed, and two molecules of 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).
上述のようなＬ−ＦＡＢＰ蛋白質を検出するための検査キットとしては、例えば、Ｌ−ＦＡＢＰ蛋白質に対する認識部位が異なる２種類の抗体を組み合わせて用いるサンドイッチＥＬＩＳＡ法を採用した検査キットが開発されている。
ここで、図１は、Ｌ−ＦＡＢＰの保存安定性を示す図であり、図１におけるグラフはＬ−ＦＡＢＰを４℃、２５℃又は３７℃で保存した場合の保存日数によるＥＬＩＳＡ測定値の変化（−８０℃保管サンプルを１００とした場合の割合（％））を示す。
サンドイッチＥＬＩＳＡ法を採用した上記検査キットにおいて、リコンビナントＬ−ＦＡＢＰ蛋白質を使用した従来の測定標準物質（標品）では、上記Ｌ−ＦＡＢＰ蛋白質を室温（２５℃）以上で長期保存すると、図１に示したように、抗体結合能が変化してしまい、ＥＬＩＳＡ法等の抗原抗体反応を利用した免疫学的手法によるＬ−ＦＡＢＰ蛋白質の正確な測定を行うことができない問題があった。
このようにＬ−ＦＡＢＰ蛋白質の不安定性のため、Ｌ−ＦＡＢＰ蛋白質標品の製造条件や測定環境に厳密な管理が求められ、また標品は低温保存が要求され、更なる操作性、安定性の向上が望まれていた。
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.
本発明は、上記事情に鑑みてなされたものであり、幅広い保存条件において、特異的に結合する抗体による結合能の安定性に優れる肝型脂肪酸結合蛋白質、該蛋白質をコードするＤＮＡ、該ＤＮＡで形質転換された細胞、該蛋白質の製造方法、該蛋白質を用いた標品、該蛋白質の検量線を作成する方法、及び該蛋白質を定量する方法の提供を目的とする。 The present invention has been made in view of the above circumstances, and has a liver-type fatty acid binding protein excellent in stability of binding ability by an antibody that specifically binds under a wide range of storage conditions, a DNA encoding the protein, and the DNA. It is an object of the present invention to provide transformed cells, a method for producing the protein, a preparation using the protein, a method for preparing a calibration curve for the protein, and a method for quantifying the protein.
本発明者らは、上述した問題点を解決すべく鋭意検討した結果、Ｌ−ＦＡＢＰ蛋白質は酸化剤である２，２’−アゾビス２−アミジノプロパン（以下、ＡＡＰＨと略記）処理によってメチオニン残基の酸化修飾及び構造変化が生じ、その結果、ＥＬＩＳＡにおける抗体結合能が変化し、ＥＬＩＳＡ測定値が大きく変動することを見出した。また、驚くべきことに、ＡＡＰＨ無添加の場合（Ｈ２Ｏを添加）においても室温で１時間反応させることによりＥＬＩＳＡ測定値が上昇しており、空気酸化による影響が生じていることも見出した。 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. .
更に、Ｌ−ＦＡＢＰ蛋白質中のメチオニン残基を遺伝子改変技術により別のアミノ酸に変異させたＬ−ＦＡＢＰ蛋白質が、酸化反応や脂肪酸の添加、室温以上の温度における長期保存においても抗体結合能が変化せずに安定化されることを見出した。
本発明は、上記知見に基づき完成されるに至ったものである。
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.
すなわち本発明は以下の通りである。
本発明の第１の態様は、
配列表の配列番号１と相同性９０％以上のアミノ酸配列からなり、１９番目、７４番目、１１３番目の１つ以上のメチオニンが、メチオニン以外の非極性アミノ酸に置換された肝型脂肪酸結合蛋白質である。
本発明の第２の態様は、
上記第１の態様に係る蛋白質をコードするＤＮＡである。
本発明の第３の態様は、
上記第２の態様に係るＤＮＡで形質転換された細胞である。
本発明の第４の態様は、
上記第３の態様に係る細胞を培養し、上記第１の態様に係る蛋白質を回収する工程を含む上記第１の態様に係る蛋白質の製造方法である。
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 homology with SEQ ID NO: 1 in the sequence listing, wherein one or more methionines at the 19th, 74th, and 113th positions are substituted with nonpolar amino acids other than methionine. is there.
The second aspect of the present invention is:
DNA encoding the protein according to the first aspect.
The third aspect of the present invention is:
A cell transformed with the DNA according to the second aspect.
The fourth aspect of the present invention is:
A method for producing a protein according to the first aspect, comprising the steps of culturing the cell according to the third aspect and recovering the protein according to the first aspect.
本発明の第５の態様は、
上記第１の態様に係る蛋白質からなる肝型脂肪酸結合蛋白質標品である。
本発明の第６の態様は、
配列表の配列番号１と相同性９０％以上のアミノ酸配列からなり、１９番目、７４番目、１１３番目の１つ以上はメチオニンであり、前記メチオニンの少なくとも１つが６６．７％超の酸化率を有する肝型脂肪酸結合蛋白質標品である。
本発明の第７の態様は、
アラキドン酸、オレイン酸、８−イソプロスタグランジンＦ２α及び２，３−ジノル−８−イソプロスタグランジンＦ２αよりなる群から選択される少なくとも１種の脂肪酸を０．３５μＭ以上含み、かつ配列表の配列番号１と相同性９０％以上のアミノ酸配列からなる肝型脂肪酸結合蛋白質を含む肝型脂肪酸結合蛋白質標品である。
According to a fifth aspect of the present invention,
A liver type fatty acid binding protein preparation comprising the protein according to the first aspect.
The sixth aspect of the present invention is:
It consists of an amino acid sequence having a homology of 90% or more with SEQ ID NO: 1 in the sequence listing, and one or more of the 19th, 74th and 113th are methionine, and at least one of the methionine has an oxidation rate of more than 66.7%. It is a liver type fatty acid binding protein preparation.
The seventh aspect of the present invention is
0.35 μM or more of 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 It is a liver type fatty acid binding protein preparation containing a liver type fatty acid binding protein consisting of an amino acid sequence having a homology of 90% or more with SEQ ID NO: 1 in the table.
本発明の第８の態様は、
上記第５〜７のいずれかの態様に係る肝型脂肪酸結合蛋白質標品を用いて、肝型脂肪酸結合蛋白質の検量線を作成する方法である。
本発明の第９の態様は、
上記第８の態様に係る方法で作成した検量線を用いて、試料中の肝型脂肪酸結合蛋白質を定量する方法である。
The eighth 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 any one of the fifth to seventh aspects.
The ninth aspect of the present invention provides
This 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 eighth aspect.
本発明によれば、幅広い保存条件において、特異的に結合する抗体による結合能の安定性に優れる肝型脂肪酸結合蛋白質、該蛋白質をコードするＤＮＡ、該ＤＮＡで形質転換された細胞、該蛋白質の製造方法、該蛋白質を用いた標品、該蛋白質の検量線を作成する方法、及び該蛋白質を定量する方法を提供することができる。
本発明の肝型脂肪酸結合蛋白質の製造方法は、得られる肝型脂肪酸結合蛋白質が酸化や脂肪酸結合状態による抗体結合能の変化を抑制することができることから、由来生物種や蛋白質発現方法によらない肝型脂肪酸結合蛋白質標品を提供することができる。すなわち肝型脂肪酸結合蛋白質標品の製造条件、保存条件、操作性における安定性と汎用性を提供することができる。
According to the present invention, a liver type fatty acid-binding protein excellent in stability of binding ability by an antibody that specifically binds under a wide range of storage conditions, DNA encoding the protein, cells transformed with the DNA, A production method, a preparation using the protein, a method for preparing a calibration curve for the protein, and a method for quantifying the protein can be provided.
The method for producing a liver-type fatty acid binding protein of the present invention does not depend on the source species or protein expression method because the obtained liver-type fatty acid binding protein can suppress changes in antibody binding ability due to oxidation or fatty acid binding state. A liver-type fatty acid binding protein preparation can be provided. That is, it is possible to provide stability and versatility in the production conditions, storage conditions, and operability of the liver type fatty acid binding protein preparation.
本発明の肝型脂肪酸結合蛋白質標品は、上記安定性に優れる肝型脂肪酸結合蛋白質からなるので、温度や酸化の程度によらずに、正確な検出ないし定量を可能とする。
また、本発明の肝型脂肪酸結合蛋白質標品は、幅広い保存条件において、特異的に結合する抗体による結合能の安定性に優れることから、測定用標準物質又は精度管理用物質としての肝型脂肪酸結合蛋白質の製造管理を容易にし、免疫学的手法における測定の操作性、汎用性が向上することが期待される。
Since the liver-type fatty acid binding protein preparation of the present invention comprises the above-described liver-type fatty acid binding protein having excellent stability, accurate detection or quantification is possible regardless of the temperature and the degree of oxidation.
In addition, since the liver type fatty acid binding protein preparation of the present invention is excellent in stability of binding ability by antibodies that specifically bind under a wide range of storage conditions, liver type fatty acid as a measurement standard substance or a quality control substance It is expected that the production management of the binding protein is facilitated, and the operability and versatility of the measurement in the immunological technique are improved.
以下、本発明の実施態様について詳細に説明するが、本発明は、以下の実施態様に何ら限定されるものではなく、本発明の目的の範囲内において、適宜変更を加えて実施することができる。 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. .
＜安定化された肝型脂肪酸結合蛋白質＞
本発明の第１の態様に係る肝型脂肪酸結合蛋白質は、
配列表の配列番号１と相同性９０％以上のアミノ酸配列からなり、１９番目、７４番目、１１３番目の１つ以上のメチオニンが、メチオニン以外の非極性アミノ酸に置換された変異肝型脂肪酸結合蛋白質である。
第１の態様に係る肝型脂肪酸結合蛋白質は、１９番目、７４番目、１１３番目の１つ以上のメチオニンがメチオニン以外の非極性アミノ酸に置換されることにより、肝型脂肪酸結合蛋白質に対する特異的に結合する抗体の結合能を安定化することができる。
メチオニン以外にも、ジスルフィド結合や酸素の直接付加を受けるシステイン残基や、カルボニル化するリシン残基、アルギニン残基、プロリン残基、ニトロ化されるチロシン残基等の酸化修飾が知られているが（Ｔｏｄａ Ｔ．，ｅｔａｌ．，基礎老化研究，３５（３）；１７−２２，２０１１）、１９番目、７４番目、１１３番目の１つ以上のメチオニンを置換するアミノ酸が非極性アミノ酸であることにより酸化修飾を防ぐことができる。
<Stabilized liver-type fatty acid binding protein>
The liver-type fatty acid binding protein according to the first aspect of the present invention is:
A mutant liver-type fatty acid binding protein comprising an amino acid sequence having a homology of 90% or more with SEQ ID NO: 1 in the sequence listing, wherein one or more methionines at the 19th, 74th, and 113th positions are replaced with nonpolar amino acids other than methionine It is.
The liver-type fatty acid binding protein according to the first aspect is specifically directed to the liver-type fatty acid binding protein by replacing one or more methionines at the 19th, 74th, and 113th with nonpolar amino acids other than methionine. The binding ability of the antibody to be bound 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.
配列番号１は、野生型ヒトＬ−ＦＡＢＰ蛋白質（以下、Ｌ−ＦＡＢＰ ＷＴともいう。）のアミノ酸配列を表す。
本明細書で言う「相同性９０％以上のアミノ酸配列」とは、アミノ酸の相同性が９０％以上であることを意味し、相同性は好ましくは９５％以上、より好ましくは９７％以上である。
配列表の配列番号１に記載した野生型ヒト肝型脂肪酸結合蛋白質のアミノ酸配列上の置換、挿入、欠失等による変異蛋白質であっても、その変異が野生型ヒト肝型脂肪酸結合蛋白質の３次元構造において保存性が高い変異であれば、これらは全て肝型脂肪酸結合蛋白質の範囲内に属し得る。
蛋白質の構成要素となるアミノ酸の側鎖は、疎水性、電荷、大きさなどにおいてそれぞれ異なるものであるが、実質的にタンパク質全体の３次元構造（立体構造とも言う）に影響を与えないという意味で保存性の高い幾つかの関係が、経験的にまた物理化学的な実測により知られている。例えば、アミノ酸残基の置換については、グリシン（Ｇｌｙ）とプロリン（Ｐｒｏ）、Ｇｌｙとアラニン（Ａｌａ）又はバリン（Ｖａｌ）、ロイシン（Ｌｅｕ）とイソロイシン（Ｉｌｅ）、グルタミン酸（Ｇｌｕ）とグルタミン（Ｇｌｎ）、アスパラギン酸（Ａｓｐ）とアスパラギン（Ａｓｎ）、システイン（Ｃｙｓ）とスレオニン（Ｔｈｒ）、Ｔｈｒとセリン（Ｓｅｒ）又はＡｌａ、リジン（Ｌｙｓ）とアルギニン（Ａｒｇ）等が挙げられる。
SEQ ID NO: 1 represents the amino acid sequence of wild-type human L-FABP protein (hereinafter also referred to as L-FABP WT).
The term “amino acid sequence having 90% or more homology” as used herein means that amino acid homology is 90% or more, and the homology 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.
上記第１の態様に係る肝型脂肪酸結合蛋白質の取得方法については特に制限はなく、化学合成により合成した蛋白質でもよいし、遺伝子組み換え技術による作製した組み換え蛋白質でもよい。
Ｌ−ＦＡＢＰ蛋白質のアミノ酸配列や遺伝子配列は既に報告されているため（Ｖｅｅｒｋａｍｐ ａｎｄ Ｍａａｔｍａｎ， Ｐｒｏｇ． Ｌｉｐｉｄ Ｒｅｓ．，３４：１７−５２，１９９５）、例えば、それらを基にプライマーを設計し、ＰＣＲ法により適当なｃＤＮＡライブラリ等からｃＤＮＡをクローニングし、これを用いて遺伝子組換えを行うことより、上記第１の態様に係る肝型脂肪酸結合蛋白質を調製することができる。
The method for obtaining the liver-type fatty acid binding protein according to the first aspect 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 liver type fatty acid binding protein according to the first aspect can be prepared by cloning cDNA from an appropriate cDNA library and carrying out gene recombination using the cDNA.
第１の態様に係る肝型脂肪酸結合蛋白質は、１９番目、７４番目、１１３番目の２つ以上のメチオニンがメチオニン以外の非極性アミノ酸に置換されることが好ましく、１９番目のメチオニンを含む２つ以上のメチオニンがメチオニン以外の非極性アミノ酸に置換されることがより好ましく、１９番目、７４番目、１１３番目のメチオニン全てがメチオニン以外の非極性アミノ酸に置換されることが更に好ましい。 In the liver-type fatty acid binding protein according to the first aspect, 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 containing 19th methionine More preferably, the above methionine is substituted with a nonpolar amino acid other than methionine, and it is further preferred that all of the 19th, 74th and 113th methionines are substituted with nonpolar amino acids other than methionine.
１９番目、７４番目、１１３番目の１つ以上のメチオニンを置換する非極性アミノ酸としては、１９番目、７４番目、１１３番目の１つ以上のメチオニンが同一の非極性アミノ酸で置換されてもよく、異なる非極性アミノ酸で置換されていてもよい。１９番目、７４番目、１１３番目の１つ以上のメチオニンを置換する非極性アミノ酸としては、ロイシン、イソロイシン、バリン、アラニン、フェニルアラニン、トリプトファンであることが好ましく、抗体の結合性を大きく変化させないメチオニンと類似した構造である観点から、ロイシン、イソロイシン、バリン、アラニンであることがより好ましく、ロイシン、イソロイシン、バリンであることが更に好ましく、ロイシン、イソロイシンであることが特に好ましく、ロイシンであることが最も好ましい。 As the nonpolar amino acid that replaces 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 the binding property of the antibody. 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.
＜安定化された肝型脂肪酸結合蛋白質をコードするＤＮＡ＞
本発明の第２の態様に係るＤＮＡは、上記第１の態様に係る変異肝型脂肪酸結合蛋白質をコードするＤＮＡである。
上記第１の態様に係る変異肝型脂肪酸結合蛋白質をコードするＤＮＡ（変異遺伝子）は、化学合成、遺伝子工学的手法又は突然変異誘発などの任意の方法で作製することができる。上述のように、Ｌ−ＦＡＢＰ蛋白質のアミノ酸配列や遺伝子配列は既に報告されているため、例えば、それらを基にプライマーを設計し、ＰＣＲ法により適当なｃＤＮＡライブラリ等からｃＤＮＡをクローニングし、これを用いて遺伝子組換えにより得ることができる。遺伝子工学的手法の一つである部位特異的変異誘発法は特定の位置に特定の変異を導入できる手法であることから有用であり、モレキュラークローニング第２版、カレント・プロトコールズ・イン・モレキュラー・バイオロジー等に記載の方法に準じて行うことができる。
<DNA encoding stabilized liver-type fatty acid binding protein>
The DNA according to the second aspect of the present invention is a DNA encoding the mutant liver-type fatty acid binding protein according to the first aspect.
The DNA (mutant gene) encoding the mutant liver-type fatty acid binding protein according to the first aspect 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.
＜形質転換細胞＞
本発明の第３の態様に係る細胞は、上記第２の態様に係るＤＮＡで形質転換された細胞である。
上記第２の態様に係るＤＮＡ又は上記第２の態様に係るＤＮＡ含む組み換えベクターを適当な宿主に導入することによって上記第３の態様に係る形質転換細胞を作製することができる。
上記第２の態様に係るＤＮＡ含む組み換えベクターは、適当な宿主ベクター系による一般的遺伝子組み換え技術によって調製することができる。適当なベクターとしては、大腸菌由来のプラスミド（例、ｐＢＲ３２２、ｐＵＣ１１８その他）、枯草菌由来のプラスミド（例、ｐＵＢ１１０、ｐＳＨ１９その他）、さらにバクテリオファージやレトロウイルスやワクシニアウイルス等の動物ウイルス等が利用できる。
上記第２の態様に係るＤＮＡ又は上記第２の態様に係るＤＮＡ含む組み換えベクターを導入される宿主細胞は、細菌、酵母等が挙げられる。
細菌細胞の例としては、コリネバクテリウム属細菌（例えば、Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕｍ）、バチルス属細菌（例えば、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）又はストレプトマイセス属細菌等のグラム陽性菌又は大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）等のグラム陰性菌が挙げられる。これら細菌の形質転換は、プロトプラスト法、または公知の方法でコンピテント細胞を用いることにより行えばよい。
酵母細胞の例としては、サッカロマイセス又はシゾサッカロマイセスに属する細胞が挙げられ、例えば、サッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｌａｅ）またはサッカロマイセス・クルイベリ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｋｌｕｙｖｅｒｉ）等が挙げられる。酵母宿主への組み換えベクターの導入方法としては、例えば、エレクトロポレーション法、スフェロブラスト法、酢酸リチウム法等を挙げることができる。
上記第２の態様に係るＤＮＡで形質転換された細胞としては、上記第１の態様に係る肝型脂肪酸結合蛋白質を効率よく製造し、かつ後述の肝型脂肪酸結合蛋白質の精製が煩雑な工程を要することがないことから、Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕｍを用いたタンパク質分泌発現系（ＣＯＲＹＮＥＸ（登録商標）：味の素株式会社製）であることが好ましい。
<Transformed cells>
The cell according to the third aspect of the present invention is a cell transformed with the DNA according to the second aspect.
The transformed cell according to the third aspect can be prepared by introducing the DNA according to the second aspect or the recombinant vector containing the DNA according to the second aspect into a suitable host.
The recombinant vector containing the DNA according to the second aspect 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 host cells into which the DNA according to the second aspect or the recombinant vector containing the DNA according to the second aspect is introduced include bacteria and yeasts.
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.
The cells transformed with the DNA according to the second aspect include a step of efficiently producing the liver fatty acid binding protein according to the first aspect and complicated purification of the liver fatty acid binding protein described later. Since it is not necessary, a protein secretion expression system (CORYNEX (registered trademark): manufactured by Ajinomoto Co., Inc.) using Corynebacterium glutamicum is preferable.
＜安定化された肝型脂肪酸結合蛋白質の製造方法＞
本発明の第４の態様に係る蛋白質の製造方法は、上記第３の態様に係る形質転換細胞を培養し、上記第１の態様に係る蛋白質を回収する工程を含む蛋白質の製造方法である。
上記第３の態様に係る形質転換細胞は、導入された遺伝子の発現を可能にする条件下で適切な栄養培地中で培養する。上記第３の態様に係る形質転換細胞の培養物から、上記第１の態様に係る蛋白質を回収するには、通常の蛋白質の単離、精製法を用いればよい。
例えば、上記第１の態様に係る蛋白質が、細胞内に溶解状態で発現した場合には、培養終了後、細胞を遠心分離により回収し水系緩衝液に懸濁後、超音波破砕機等により細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られた上清から、通常の蛋白質の単離精製法、即ち、溶媒抽出法、硫安等による塩析法、脱塩法、有機溶媒による沈殿法、ジエチルアミノエチル（ＤＥＡＥ）セファロース等のレジンを用いた陰イオン交換クロマトグラフィー法、Ｓ−Ｓｅｐｈａｒｏｓｅ ＦＦ（ファルマシア社製）等のレジンを用いた陽イオン交換クロマトグラフィー法、ブチルセファロース、フェニルセファロース等のレジンを用いた疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、アフィニティークロマトグラフィー法、クロマトフォーカシング法、等電点電気泳動等の電気泳動法等の手法を単独あるいは組み合わせて用い、精製標品を得ることができる。
上記Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕｍを用いたタンパク質分泌発現系（ＣＯＲＹＮＥＸ（登録商標）：味の素株式会社製）を用いることにより、細胞を破砕して無細胞抽出液を得るような煩雑な工程を要することがなく、任意の遠心分離後、アニオン交換クロマトグラフィー（例えば、ＨｉＴｒａｐＱ ＦＦ５ｍＬ ＦＰＬＣカラム）による精製等により精製標品を得ることができる。
<Method for producing stabilized liver-type fatty acid binding protein>
The method for producing a protein according to the fourth aspect of the present invention is a method for producing a protein comprising the steps of culturing the transformed cell according to the third aspect and recovering the protein according to the first aspect.
The transformed cell according to the third aspect is cultured in an appropriate nutrient medium under conditions that allow expression of the introduced gene. In order to recover the protein according to the first aspect from the transformed cell culture according to the third aspect, a normal protein isolation and purification method may be used.
For example, when the protein according to the first aspect is expressed in a dissolved state in the cell, the cell is recovered by centrifugation after culturing and suspended in an aqueous buffer, and then the cell is collected by an ultrasonic crusher or the like. To obtain a cell-free 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).
＜肝型脂肪酸結合蛋白質標品＞
本発明の第５の態様に係る肝型脂肪酸結合蛋白質標品は、上記第１の態様に係る安定化された蛋白質からなる肝型脂肪酸結合蛋白質標品である。上記第１の態様に係る安定化された蛋白質からなることから、特異的に結合する抗体による結合能の変動が抑制され得る。
<Liver type fatty acid binding protein preparation>
The liver type fatty acid binding protein sample according to the fifth aspect of the present invention is a liver type fatty acid binding protein sample comprising the stabilized protein according to the first aspect. Since it consists of the stabilized protein which concerns on the said 1st aspect, the fluctuation | variation of the binding ability by the antibody which couple | bonds specifically can be suppressed.
また、本発明の第６の態様に係る肝型脂肪酸結合蛋白質標品は、配列表の配列番号１と相同性９０％以上のアミノ酸配列からなり、１９番目、７４番目、１１３番目の１つ以上はメチオニンであり、前記メチオニンの少なくとも１つが６６．７％超の酸化率を有する肝型脂肪酸結合蛋白質標品である。
１９番目、７４番目、１１３番目の１つ以上はメチオニンであり、前記メチオニンの少なくとも１つが６６．７％超の酸化率であることにより、更なる酸化率の増大が抑制され結果、特異的に結合する抗体による結合能の変動が抑制され得る。
特異的に結合する抗体による結合能の変動が更に抑制され得る観点から、上記第６の態様に係る肝型脂肪酸結合蛋白質標品は、１９番目のメチオニンの酸化率が９．３％以上、７４番目のメチオニンの酸化率が５７％超、又は１１３番目のメチオニンの酸化率が６６．７％超であることが好ましい。
上記酸化率を有する肝型脂肪酸結合蛋白質標品は、ＡＡＰＨ等の酸化剤等を用いて製造することができるし、空気酸化によって製造することもできる。
上記酸化率の測定方法としては、例えば、後述する図６（ｂ）におけるＭｅｔ１９を含むペプチド断片のＭＳスペクトル、図６（ｃ）におけるＭｅｔ７４を含むペプチド断片のＭＳスペクトル、図６（ｄ）におけるＭｅｔ１１３を含むペプチド断片のＭＳスペクトルにおける酸化無処理の場合のピークと、酸化処理後のピークとの比較から算出することができる。
The liver type fatty acid binding protein preparation according to the sixth aspect of the present invention comprises an amino acid sequence having a homology of 90% or more with SEQ ID NO: 1 in the sequence listing, and one or more of the 19th, 74th and 113th positions. Is methionine, which is a liver-type fatty acid binding protein preparation in which at least one of the methionines has an oxidation rate of more than 66.7%.
One or more of the 19th, 74th, and 113th is methionine, and at least one of the methionines has an oxidation rate of more than 66.7%. Variations in binding ability due to antibodies that bind can be suppressed.
From the viewpoint that fluctuations in binding ability due to antibodies that specifically bind can be further suppressed, the liver-type fatty acid binding protein preparation according to the sixth aspect has an oxidation rate of 19% methionine of 9.3% or more, 74 It is preferable that the oxidation rate of the 1st methionine exceeds 57% or the oxidation rate of the 113th methionine exceeds 66.7%.
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.
As the method for measuring the oxidation rate, for example, an MS spectrum of a peptide fragment containing Met19 in FIG. 6 (b) described later, an MS spectrum of a peptide fragment containing Met74 in FIG. 6 (c), and Met113 in FIG. 6 (d). 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.
また、本発明の第７の態様に係る肝型脂肪酸結合蛋白質標品は、アラキドン酸、オレイン酸、８−イソプロスタグランジンＦ２α及び２，３−ジノル−８−イソプロスタグランジンＦ２αよりなる群から選択される少なくとも１種の脂肪酸を、脂肪酸が複数種の場合は合計含有量として０．３５μＭ以上含み、かつ配列表の配列番号１と相同性９０％以上のアミノ酸配列からなる肝型脂肪酸結合蛋白質を含む肝型脂肪酸結合蛋白質標品である。
図１３を参照して後述するように、本発明者らは、Ｌ−ＦＡＢＰ蛋白質に結合する脂肪酸の種類（図１３（ｂ））や濃度によってＬ−ＦＡＢＰ蛋白質の抗体結合能が変化することを見出した。
本発明の第７の態様に係る肝型脂肪酸結合蛋白質標品は、アラキドン酸、オレイン酸、８−イソプロスタグランジンＦ２α及び２，３−ジノル−８−イソプロスタグランジンＦ２αよりなる群から選択される少なくとも１種の脂肪酸を、脂肪酸が複数種の場合は合計含有量として０．３５μＭ以上含むことにより、標品となるＬ−ＦＡＢＰ蛋白質を発現する際、又は標品として使用されるアッセイ系において、由来の異なる生物種による発現系や発現部位ないし臓器によって結合している脂肪酸の種類（例えば、アラキドン酸、オレイン酸、８−イソプロスタグランジンＦ２α及び２，３−ジノル−８−イソプロスタグランジンＦ２α等）、結合量、過酸化の程度が異なる場合であっても、特異的に結合する抗体による結合能の変動が抑制され、測定用標準物質又は精度管理用物質としての標品として機能し得る。
特異的に結合する抗体による結合能の変動が更に抑制される観点から、アラキドン酸、オレイン酸、８−イソプロスタグランジンＦ２α及び２，３−ジノル−８−イソプロスタグランジンＦ２αよりなる群から選択される少なくとも１種の脂肪酸を、脂肪酸が複数種の場合は合計含有量として０．７μＭ以上含むことが好ましく、３．５μＭ以上含むことがより好ましく、７．０μＭ以上含むことが更に好ましい。
上記少なくとも１種の脂肪酸を、脂肪酸が複数種の場合は合計含有量として０．３５μＭ以上含む上記第７の態様に係る肝型脂肪酸結合蛋白質標品は、肝型脂肪酸結合蛋白質標品を含有する液に、上記少なくとも１種の脂肪酸を脂肪酸が複数種の場合は合計含有量として０．３５μＭ以上添加すること、又はそのような含有量になるような細胞培養、蛋白質単離ないし精製条件により調製することができる。また、上記少なくとも１種の脂肪酸の含有量は、上記添加量に相当する。
In addition, the liver type fatty acid binding protein preparation according to the seventh aspect of the present invention includes arachidonic acid, oleic acid, 8-isoprostaglandin F 2α and 2,3-dinor-8-isoprostaglandin F 2α . A liver type comprising at least one fatty acid selected from the group consisting of amino acid sequences having a total content of 0.35 μM or more and a homology of 90% or more with SEQ ID NO: 1 in the sequence listing when there are a plurality of fatty acids It is a liver type fatty acid binding protein preparation containing a fatty acid binding protein.
As will be described later with reference to FIG. 13, the present inventors have found that the antibody-binding ability of the L-FABP protein varies depending on the type (FIG. 13 (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 seventh aspect of the present invention is a group consisting of arachidonic acid, oleic acid, 8-isoprostaglandin F 2α and 2,3-dinor-8-isoprostaglandin F 2α. In the case where a plurality of fatty acids are selected, the total content is 0.35 μM or more, so that the L-FABP protein serving as a standard is expressed or used as a standard. In an assay system, the type of fatty acid (for example, arachidonic acid, oleic acid, 8-isoprostaglandin F 2α and 2,3-dinol-8) bound by an expression system or an expression site or organ of a different species of origin. - iso Star prostaglandin F 2.alpha etc.), binding amount, even if the degree of peroxidation are different, the variations in binding ability due to an antibody that specifically binds is suppressed It can function as a standard as reference material for measuring or control material.
From the viewpoint of further suppressing variation in binding ability due to the antibody that specifically binds, it comprises arachidonic acid, oleic acid, 8-isoprostaglandin F 2α and 2,3-dinor-8-isoprostaglandin F 2α. When at least one fatty acid selected from the group is a plurality of fatty acids, the total content is preferably 0.7 μM or more, more preferably 3.5 μM or more, and more preferably 7.0 μM or more. preferable.
The liver-type fatty acid-binding protein preparation according to the seventh aspect, which contains at least one fatty acid as a total content of 0.35 μM or more when there are a plurality of fatty acids, contains a liver-type fatty acid-binding protein preparation. In the liquid, when at least one kind of fatty acid is used, the total content is 0.35 μM or more, or it is prepared by cell culture and protein isolation or purification conditions so as to achieve such content. can do. The content of the at least one fatty acid corresponds to the amount added.
上記第５〜７のいずれかの態様に係る肝型脂肪酸結合蛋白質標品は、実用的及び商業的に利用される観点から、販売に供される肝型脂肪酸結合蛋白質標品であることが好ましい。販売に供される肝型脂肪酸結合蛋白質標品とは、販売済みの蛋白質、具体的には販売後に長期放置された肝型脂肪酸結合蛋白質標品ではない。
上記第５〜７のいずれかの態様に係る肝型脂肪酸結合蛋白質標品は、抗原抗体反応を利用した免疫学的手法によりサンプル中の肝型脂肪酸結合蛋白質を測定するためのキットの測定用標準物質又は精度管理用物質として有用であり、Ｌ−ＦＡＢＰ蛋白質に特異的に結合する抗Ｌ−ＦＡＢＰ蛋白質抗体による特異的に結合を利用したＬ−ＦＡＢＰ蛋白質の検出ないし定量等の測定の標準物質（標品）として用いることが好ましい。
上記第５〜７のいずれかの態様に係る肝型脂肪酸結合蛋白質標品が用いられるＬ−ＦＡＢＰ蛋白質の検出ないし定量等の測定としては、酵素免疫測定法（ＥＩＡ，ＥＬＩＳＡ）、蛍光酵素免疫測定法（ＦＬＥＩＡ）、化学発光酵素免疫測定法（ＣＬＥＩＡ）、化学発光免疫測定法（ＣＬＩＡ）、電気化学発光測免疫測定法（ＥＣＬＩＡ）、イムノクロマトグラフィー法（ＩＣＡ）、ラテックス凝集法（ＬＡ）、蛍光抗体法（ＦＡ）、ラジオイムノアッセイ（ＲＩＡ）、ウェスタンブロット法（ＷＢ）、イムノブロット法などを採用したアッセイ等が挙げられ、抗原（Ｌ−ＦＡＢＰ蛋白質）に対する認識部位が異なる２種類の抗体を組み合わせて用いるサンドイッチＥＬＩＳＡ法を採用したアッセイであることが好ましい。
認識部位が異なる２種類の抗体は、一方を、マイクロプレートのウェル中の表面に結合させた固相化抗体として用い、他方を、検出ないし定量のための標識抗体として用いることが好ましい。上記標識抗体における標識としては特に制限はなく、例えば、パーオキシダーゼ標識等の酵素標識、蛍光標識、紫外線標識、放射線標識等が挙げられる。
The liver-type fatty acid binding protein preparation according to any one of the fifth to seventh aspects 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 any one of the above aspects 5 to 7 is a measurement standard for a kit for measuring liver type fatty acid binding protein in a sample by an immunological technique utilizing an antigen-antibody reaction. Standard substance for detection or quantification of L-FABP protein using specific binding by anti-L-FABP protein antibody which is useful as a substance or a substance for quality control and specifically binds to L-FABP protein ( It is preferable to use it as a standard product.
Examples of measurement such as detection or quantification of L-FABP protein in which the liver type fatty acid binding protein preparation according to any one of the fifth to seventh aspects is used include enzyme immunoassay (EIA, ELISA), fluorescent enzyme immunoassay (FLEIA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), immunochromatography (ICA), latex agglutination (LA), fluorescence Examples include assays employing antibody method (FA), radioimmunoassay (RIA), western blot method (WB), immunoblot method, etc., combining two antibodies with different recognition sites for antigen (L-FABP protein) It is preferable that the assay adopts the sandwich ELISA method used.
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.
抗原（Ｌ−ＦＡＢＰ蛋白質）に対する認識部位が異なる抗体としては、抗Ｌ−ＦＡＢＰ抗体クローン１、クローン２、クローンＬ及びクローンＦよりなる群から選択される抗体を含む抗体が挙げられ、抗Ｌ−ＦＡＢＰ抗体クローンＬを含む組み合わせ、又は抗Ｌ−ＦＡＢＰ抗体クローン２を含む組み合わせであることが好ましく、抗Ｌ−ＦＡＢＰ抗体クローンＬを含む組み合わせであることがより好ましく、抗Ｌ−ＦＡＢＰ抗体クローンＬを固相化抗体として用い、任意の抗Ｌ−ＦＡＢＰ抗体を標識抗体として用いることが更に好ましく、抗Ｌ−ＦＡＢＰ抗体クローンＬを固相化抗体として用い、抗Ｌ−ＦＡＢＰ抗体クローン２を標識抗体として用いることが特に好ましい。 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.
そのようなアッセイに用いられるＬ−ＦＡＢＰ蛋白質測定キットとしては、標品として上記第５〜７のいずれかの態様に係る肝型脂肪酸結合蛋白質標品を含み、試薬として上記抗Ｌ−ＦＡＢＰ蛋白質抗体を含むことが好ましく、標識抗Ｌ−ＦＡＢＰ蛋白質抗体を更に含むことがより好ましく、必要に応じて前処理液（任意の緩衝液、任意の界面活性剤等）、反応緩衝液（任意の緩衝液等）、発色基質（３，３’，５，５’−テトラメチルベンジジン、過酸化水素水等）等を含んでいてもよい。
Ｌ−ＦＡＢＰ蛋白質測定キットとして、抗原に対する認識部位が異なる２種類の抗体を組み合わせて用いるサンドイッチＥＬＩＳＡ法を用いたキットであることが好ましく、固相に任意の抗Ｌ−ＦＡＢＰ抗体、標識抗体に抗Ｌ−ＦＡＢＰ抗体クローン２を使用しているキットであることがより好ましい。
サンドイッチＥＬＩＳＡ法を用いたＬ−ＦＡＢＰ蛋白質測定キットの具体的態様として、例えば、下記（１）〜（９）を含むキットが挙げられる。
（１）Ｌ−ＦＡＢＰ抗体固相化マイクロプレート……抗ヒトＬ−ＦＡＢＰマウスモノクローナル抗体結合ウェル
（２）前処理液
（３）反応緩衝液
（４）酵素標識抗体……パーオキシダーゼ標識抗ヒトＬ−ＦＡＢＰマウスモノクローナル抗体〔クローン２産生細胞株由来〕
（５）酵素基質液
（６）洗浄剤（任意の緩衝液、界面活性剤等）
（７）反応停止液（１Ｎ硫酸等）
（８）標準緩衝液（任意の緩衝液等）
（９）肝型脂肪酸結合蛋白質標品
（９）肝型脂肪酸結合蛋白質標品としては、従来は、任意の緩衝液にリコンビナントヒトＬ−ＦＡＢＰを混合させた液が用いられてきたが、任意の緩衝液に上記肝型脂肪酸結合蛋白質標品を混合した液が好ましく、標品の濃度としては特に制限はなく、例えば、１０〜１００００ｎｇ／ｍＬが挙げられ、５０〜５０００ｎｇ／ｍＬが好ましく、１００〜１０００ｎｇ／ｍＬがより好ましく、２００〜８００ｎｇ／ｍＬが更に好ましく、３００〜６００ｎｇ／ｍＬが特に好ましい。
肝型脂肪酸結合蛋白質標品として、従来のリコンビナントヒトＬ−ＦＡＢＰを用いたこと以外は上記第５〜７のいずれかの態様に係る肝型脂肪酸結合蛋白質標品を用いたサンドイッチＥＬＩＳＡ法を利用したＬ−ＦＡＢＰ蛋白質測定キットと同様のキットの市販品としては、「レナプロ Ｌ−ＦＡＢＰ テスト ＴＭＢ」（シミックホールディングス社製）が挙げられる。
As an L-FABP protein measurement kit used in such an assay, the liver-type fatty acid binding protein sample according to any one of the fifth to seventh aspects is used as a preparation, and the anti-L-FABP protein antibody is used as a reagent. , More preferably a labeled anti-L-FABP protein antibody, and pretreatment liquid (arbitrary buffer, arbitrary surfactant, etc.), reaction buffer (arbitrary buffer, if necessary) Etc.), a chromogenic substrate (3,3 ′, 5,5′-tetramethylbenzidine, hydrogen peroxide solution, etc.) and the like.
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.
As a liver type fatty acid binding protein sample, a sandwich ELISA method using the liver type fatty acid binding protein sample according to any one of the above fifth to seventh aspects was used except that the conventional recombinant human L-FABP was used. “Lenapro L-FABP Test TMB” (manufactured by Simic Holdings Co., Ltd.) is an example of a commercially available kit similar to the L-FABP protein measurement kit.
安定化されたＬ−ＦＡＢＰ蛋白質からなる肝型脂肪酸結合蛋白質標品の保存溶液はタンパク吸着防止を目的としてウシ血清アルブミン（ＢＳＡ）を含有する蛋白質保存緩衝液とすることが好ましい。例えば、下記蛋白質保存緩衝液が挙げられる。
（蛋白質保存緩衝液）
１０ｍＭリン酸バッファー（ｐＨ７．２）、１５０ｍＭ ＮａＣｌ、１．０％ＢＳＡ、０．１％ＮａＮ３
The preservation solution of a liver type fatty acid binding protein preparation comprising a stabilized L-FABP protein is preferably a protein preservation buffer containing bovine serum albumin (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
＜検量線を作成する方法及び肝型脂肪酸結合蛋白質を定量する方法＞
本発明の第８の態様に係る肝型脂肪酸結合蛋白質の検量線を作成する方法は、上記第５〜７のいずれかの態様に係る肝型脂肪酸結合蛋白質標品を用いる。
具体的には、測定される上記標識の強度（例えば、酵素標識強度、蛍光強度、紫外線強度、放射線強度等）と、上記標品の量（例えば、濃度）との関係に基づき検量線を作成することができる。
本発明の第９の態様に係る試料中の肝型脂肪酸結合蛋白質を定量する方法は、上記第８の態様に係る方法で作成した検量線を用いる。
具体的には、上記検量線の作成と同様な条件により、標識抗体等により標識された試料中の肝型脂肪酸結合蛋白質の標識強度を測定し、上記検量線に基づき（例えば、対比し）、試料中の肝型脂肪酸結合蛋白質を検出ないし定量することができる。
<Method for preparing calibration curve and method for quantifying liver-type fatty acid binding protein>
The method for preparing a calibration curve for liver-type fatty acid binding protein according to the eighth aspect of the present invention uses the liver-type fatty acid binding protein preparation according to any one of the above fifth to seventh aspects.
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 a sample according to the ninth aspect of the present invention uses the calibration curve created by the method according to the eighth 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.
以下、実施例を示して本発明を更に詳細に説明するが、本発明の範囲は、これらの実施例に限定されるものではない。 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.
参考例
＜酸化によるＬ−ＦＡＢＰ蛋白質の構造変化＞
Ｌ−ＦＡＢＰ蛋白質をＡＡＰＨ処理した。結果を図２（ａ）及び（ｂ）に示す。
図２（ａ）は酸化型Ｌ−ＦＡＢＰ及び非酸化型Ｌ−ＦＡＢＰのＬＣ−ＥＳＩ−ＭＳによる分子量測定結果を示す図であり、図２（ｂ）は酸化型Ｌ−ＦＡＢＰと非酸化型Ｌ−ＦＡＢＰのＣＤスペクトルを示す図である。
図２（ａ）から明らかなように、酸化型Ｌ−ＦＡＢＰでは理論分子量よりおおよそ酸素分子２〜３分子に相当する分子量増加が観測されている。
図２（ｂ）に示したように、酸化型Ｌ−ＦＡＢＰでは３５０ｎｍ付近（図中矢印）に蛍光ピークが出現しており、Ｌ−ＦＡＢＰ蛋白質中に存在する芳香族アミノ酸周辺が極性の高い環境に変化したことを意味しており、酸化型Ｌ−ＦＡＢＰと非酸化型Ｌ−ＦＡＢＰでは構造変化を生じていると考えられる。
Reference Example <Structural 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 diagram showing the molecular weight measurement results by LC-ESI-MS of oxidized L-FABP and non-oxidized L-FABP, and FIG. 2 (b) shows oxidized L-FABP and non-oxidized L FIG. 6 shows a CD spectrum of FABP.
As is clear from FIG. 2A, in the oxidized L-FABP, an increase in molecular weight corresponding to about 2 to 3 oxygen molecules is observed from the theoretical molecular weight.
As shown in FIG. 2B, in the oxidized L-FABP, a fluorescent peak appears around 350 nm (arrow in the figure), and the environment around the aromatic amino acid present in the L-FABP protein is highly polar. It is considered that the structural change occurred between oxidized L-FABP and non-oxidized L-FABP.
＜酸化によるＥＬＩＳＡ測定値の変化＞
ＡＡＰＨを所定の濃度になるようＬ−ＦＡＢＰ蛋白質溶液に添加し、室温で１時間反応させ、ＥＬＩＳＡ測定を行った。結果を図３に示す。
図３から、Ｌ−ＦＡＢＰ蛋白質のＡＡＰＨ処理によりＥＬＩＳＡ測定値の変化（無処理を１００とした場合の割合（％））が生じていることがわかる。
また、驚くべきことに、図３に示したように、ＡＡＰＨ無添加サンプル（Ｈ２Ｏを添加）においても室温で１時間反応させることによりＥＬＩＳＡ測定値が上昇しており、空気酸化による影響が生じていると考えられる。
<Change in ELISA measurement value due to oxidation>
AAPH was added to the L-FABP protein solution to a predetermined concentration and reacted at room temperature for 1 hour, and ELISA measurement was performed. The results are shown in FIG.
From FIG. 3, it can be seen that the change in the ELISA measurement value (ratio (%) when no treatment is taken as 100) is caused by the AAPH treatment of the L-FABP protein.
Surprisingly, as shown in FIG. 3, even in the AAPH-free sample (added with H 2 O), the ELISA measurement value was increased by reacting at room temperature for 1 hour, and the influence of air oxidation was affected. It is thought that it has occurred.
＜ヒトＬ−ＦＡＢＰ蛋白質の酸化＞
各種濃度（４０ｍＭ、２００ｍＭ）のＡＡＰＨをヒトＬ−ＦＡＢＰ蛋白質に添加し、３７℃で１．５時間反応させた。結果を図４に示す。
図４は、各種濃度（４０ｍＭ、２００ｍＭ）のＡＡＰＨをヒトＬ−ＦＡＢＰ蛋白質に添加し、３７℃で１．５時間反応させた後のＭＳスペクトルを示す図である。
図４に示したように、ＡＡＰＨ処理によりＬ−ＦＡＢＰに酸素分子１〜３分子に相当する分子量増加が観測された。
<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 due to AAPH treatment.
＜ヒトＬ−ＦＡＢＰ蛋白質が含有するメチオニンの酸化＞
図５（ａ）は、配列番号１に示したヒトＬ−ＦＡＢＰ蛋白質のアミノ酸配列を示す図であり、図５（ｂ）は、ヒトＬ−ＦＡＢＰ蛋白質のトリプシン消化によって生じ得る各種ペプチド断片の推定分子量を示す図である。
上記各種濃度（４０ｍＭ、２００ｍＭ）のＡＡＰＨにより反応した後のヒトＬ−ＦＡＢＰ蛋白質をトリプシン消化して得られた各種ペプチド断片のＭＳスペクトルを測定した。
図６（ａ）は、上記各種濃度（４０ｍＭ、２００ｍＭ）のＡＡＰＨにより反応した後のヒトＬ−ＦＡＢＰ蛋白質をトリプシン消化して得られたＭｅｔ１を含むペプチド断片Ｎｏ．１のＭＳスペクトルを示す図であり、図６（ｂ）は、上記各種濃度のＡＡＰＨと反応後のヒトＬ−ＦＡＢＰ蛋白質をトリプシン消化して得られたＭｅｔ１９を含むペプチド断片Ｎｏ．２のＭＳスペクトルを示す図であり、図６（ｃ）は、上記各種濃度のＡＡＰＨと反応後のヒトＬ−ＦＡＢＰ蛋白質をトリプシン消化して得られたＭｅｔ７４を含むペプチド断片Ｎｏ．９及びペプチド断片Ｎｏ．１０のＭＳスペクトルを示す図であり、図６（ｄ）は、上記各種濃度のＡＡＰＨと反応後のヒトＬ−ＦＡＢＰ蛋白質をトリプシン消化して得られたＭｅｔ１１３を含むペプチド断片Ｎｏ．１４及びペプチド断片１５のＭＳスペクトルを示す図である。
図６（ａ）〜（ｄ）に示した結果から、酸化修飾部位は１９番目、７４番目、１１３番目のメチオニン残基（以下、それぞれＭｅｔ１９、Ｍｅｔ７４、Ｍｅｔ１１３ともいう。）であることが明らかになった。
<Oxidation of methionine contained in human L-FABP protein>
FIG. 5 (a) shows the amino acid sequence of the human L-FABP protein shown in SEQ ID NO: 1, and FIG. 5 (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. 6A 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. 6 (b) is a peptide fragment No. 1 containing Met19 obtained by trypsin digesting the human L-FABP protein after the reaction with the above-mentioned various concentrations of AAPH. FIG. 6 (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. 6 (d) shows the peptide fragment No. 10 containing Met113 obtained by trypsin digestion of the human L-FABP protein after the 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.
実施例１
＜変異Ｌ−ＦＡＢＰ蛋白質の製造方法＞
Ｍｅｔ１９をロイシン残基に変異させた変異蛋白質（以下、Ｌ−ＦＡＢＰ Ｍ１９Ｌともいう。）、
Ｍｅｔ７４をロイシン残基に変異させた変異蛋白質（以下、Ｌ−ＦＡＢＰ Ｍ７４Ｌともいう。）、
Ｍｅｔ１１３をロイシン残基に変異させた変異蛋白質（以下、Ｌ−ＦＡＢＰ Ｍ１１３Ｌともいう。）、
Ｍｅｔ１９及びＭｅｔ７４をそれぞれロイシン残基に変異させた変異蛋白質（以下、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌともいう。）、
Ｍｅｔ１９及びＭｅｔ１１３をそれぞれロイシン残基に変異させた変異蛋白質（以下、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ１１３Ｌともいう。）、
Ｍｅｔ７４及びＭｅｔ１１３をそれぞれロイシン残基に変異させた変異蛋白質（以下、Ｌ−ＦＡＢＰ Ｍ７４Ｌ／Ｍ１１３Ｌともいう。）、
３つのメチオニン残基（Ｍｅｔ１９、Ｍｅｔ７４、Ｍｅｔ１１３）をそれぞれロイシン残基に変異させた変異蛋白質（以下、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌともいう。）を製造した。
Ｌ−ＦＡＢＰ Ｍ１９Ｌ、
Ｌ−ＦＡＢＰ Ｍ７４Ｌ、
Ｌ−ＦＡＢＰ Ｍ１１３Ｌ、
Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ、
Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ１１３Ｌ、
Ｌ−ＦＡＢＰ Ｍ７４Ｌ／Ｍ１１３Ｌ、及び
Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌは、それぞれ、グラム陽性細菌Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕｍを用いたタンパク質分泌発現系（ＣＯＲＹＮＥＸ（登録商標）：味の素株式会社製）を利用して発現を行った。
Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌのアミノ酸配列を後記配列表の配列番号２に示した。
蛋白質発現に用いたＬ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌの遺伝子配列を後記配列表の配列番号３に示した。
Example 1
<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.
上述した方法により発現を行ったＬ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌ等は、まず遠心式フィルターユニット Ａｍｉｃｏｎ（登録商標）Ｕｌｔｒａ−１５、３ｋＤａ（ミリポア社製）を用いバッファーＡ（１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．５），１ｍＭ ＤＴＴ）とバッファー交換を行った。
次に、ＨｉＴｒａｐＱ ＦＦカラム、５ｍＬ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）を用い精製を行った。ＨｉＴｒａｐＱ ＦＦカラムに吸着したＬ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌは、バッファーＡで洗浄後、バッファーＡとバッファーＢ（１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．５），１ｍＭ ＤＴＴ，２Ｍ ＮａＣｌ）による直線的濃度勾配法により溶出させ、バッファーＢが３．１％となるピークを含むフラクションを回収した。回収した蛋白質に関してはＳＤＳ−ＰＡＧＥ及びＳｉｌｖｅｒ ＳｔａｉｎＩＩ Ｋｉｔ Ｗａｋｏ（和光純薬株式会社製）による蛋白質染色を行い、精製度合いの確認を行った。Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌについての精製結果を図７に示す。
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. The purification results for L-FABP M19L / M74L / M113L are shown in FIG.
上述の操作により得たＬ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌ等は−８０℃にて保管した。 L-FABP M19L / M74L / M113L obtained by the above operation was stored at -80 ° C.
ＡＡＰＨを所定の最終濃度（０ｍＭ、０．５ｍＭ、１ｍＭ、２ｍＭ、４ｍＭ）になるようＬ−ＦＡＢＰ蛋白質溶液（Ｌ−ＦＡＢＰ Ｍ１９Ｌ、Ｌ−ＦＡＢＰ Ｍ７４Ｌ、Ｌ−ＦＡＢＰ Ｍ１１３Ｌ、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ１１３Ｌ、Ｌ−ＦＡＢＰ Ｍ７４Ｌ／Ｍ１１３Ｌ、及びＬ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌの各溶液）に添加し、室温で１時間反応させ、ＥＬＩＳＡ測定を実施し、標識抗体の発色（ＯＤ４５０ｎｍ）を無処理サンプルと比較した。比較結果を図８に示す。
図８に示した結果から明らかなように、１９番目のメチオニンのロイシンへの置換が酸化に対する安定性に寄与が大きいことが分かる（Ｌ−ＦＡＢＰ Ｍ１９Ｌ）。
特に、１９番目のメチオニンを含む２つ以上のメチオニンのロイシンへの置換が酸化に対する安定性に寄与が特に大きいことが分かる（Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ１１３Ｌ）。
Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌが最も酸化に対する安定性に優れることが分かる。
次に、酸化安定性を確認したＬ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌ等はサンプルの長期保存のため上述した蛋白質保存緩衝液に溶解し、−８０℃にて保管し、以下実施例２〜６に使用した。
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. 8, it can be seen that the substitution of the 19th methionine with leucine greatly contributes to the stability against oxidation (L-FABP M19L).
In particular, it can be seen that substitution of two or more methionines, including the 19th methionine, with leucine has a particularly large contribution to the stability against oxidation (L-FABP M19L / M74L, L-FABP M19L / M113L).
It can be seen that L-FABP M19L / M74L / M113L is most excellent in stability against oxidation.
Next, L-FABP M19L / M74L / M113L and the like whose oxidation stability was confirmed were dissolved in the protein storage buffer described above for long-term storage of the sample and stored at −80 ° C. used.
実施例２
＜酸化による安定性＞
Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌに対してＡＡＰＨを最終濃度０〜５ｍＭとなるよう添加し、室温で１時間反応させた。また空気酸化の影響を考慮し、蛋白質溶液にＨ２Ｏのみを添加し、添加直後に測定を実施した無処理サンプルを比較対象とした。
Example 2
<Stability by 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.
これらの反応溶液及び無処理サンプルを「レナプロ Ｌ−ＦＡＢＰ テスト ＴＭＢ」（シミックホールディングス株式会社製）を使用してＥＬＩＳＡ測定を実施し、標識抗体の発色（ＯＤ４５０ｎｍ）を無処理サンプルと比較した。上記診断用キットの使用方法は通常添付されている添付文書に従った測定方法に準じて行った。結果を図９に示す。 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.
図９に示したＥＬＩＳＡ測定の結果から明らかなように、Ｌ−ＦＡＢＰ ＷＴは１１２〜１２１％の測定値の上昇が確認され、ばらつきを表す変動係数（ＣＶ）は５．８％であった。
一方、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌは１０２〜１０７％の上昇までしか認められず、ＣＶは２．４％であった。
以上の結果からＡＡＰＨに対してＬ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌの抗体結合能は安定的であり、ＥＬＩＳＡ測定値の変動は小さいことが明らかとなった。
つまり、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌは酸化反応に対して安定化したといえる。また室温に１時間置くだけでもＬ−ＦＡＢＰ ＷＴの抗体結合能が上昇するが、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌは上昇しないことから、空気酸化に対しても安定化したといえる。
As is clear from the results of the ELISA measurement shown in FIG. 9, 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.
実施例３
＜室温での安定性＞
Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌを室温（２５℃）にて保存し、１週間ごとに４週間後まで「レナプロ Ｌ−ＦＡＢＰ テスト ＴＭＢ」を使用して定法に従いＥＬＩＳＡ測定を実施した。−８０℃にて保存しているサンプルに関しても測定を実施し、比較対象とした。室温保存サンプルの標識抗体の発色（ＯＤ４５０ｎｍ）について−８０℃保存サンプルの標識抗体の発色（ＯＤ４５０ｎｍ）を１００とした割合（％）を比較した。結果を図１０に示す。
Example 3
<Stability at room temperature>
L-FABP M19L / M74L / M113L was stored at room temperature (25 ° C.), and ELISA measurement was performed according to a conventional method using “Lenapro L-FABP Test TMB” every week until 4 weeks later. Measurements were also performed on samples stored at −80 ° C. and used as comparison targets. Regarding the color development (OD450 nm) of the labeled antibody of the room temperature storage sample, the ratio (%) where the color development (OD450 nm) of the label antibody of the −80 ° C. storage sample was 100 was compared. The results are shown in FIG.
図１０に示したＥＬＩＳＡ測定の結果から明らかなように、Ｌ−ＦＡＢＰ ＷＴは１１０〜１２８％の測定値の上昇が確認され、ＣＶは１０．５％であった。
一方、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌは１０１〜１１１％の上昇までしか認められず、ＣＶは４．７％であった。
以上の結果から室温での長期保存に対してＬ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌの抗体結合能は安定的であり、ＥＬＩＳＡ測定値の変動は小さいことが明らかとなった。つまり、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌは室温での長期保存に安定化されたといえる。
As is clear from the results of the ELISA measurement shown in FIG. 10, the L-FABP WT was confirmed to have an increase in the measured value of 110 to 128%, and the 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 and the fluctuation of the ELISA measurement value is small with respect to long-term storage at room temperature. That is, it can be said that L-FABP M19L / M74L / M113L has been stabilized for long-term storage at room temperature.
実施例４
＜３７℃での安定性＞
Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌを３７℃で保存し、１週間ごとに４週間後まで「レナプロ Ｌ−ＦＡＢＰ テスト ＴＭＢ」を使用して定法に従いＥＬＩＳＡ測定を実施した。−８０℃にて保存しているサンプルに関しても測定を実施し、比較対象とした。室温保存サンプルの標識抗体の発色（ＯＤ４５０ｎｍ）について−８０℃保存サンプルの標識抗体の発色（ＯＤ４５０ｎｍ）を１００とした割合（％）を比較した。結果を図１１に示す。
Example 4
<Stability at 37 ° C>
L-FABP M19L / M74L / M113L was stored at 37 ° C., and ELISA measurement was performed according to a conventional method using “Lenapro L-FABP Test TMB” every week until 4 weeks later. Measurements were also performed on samples stored at −80 ° C. and used as comparison targets. Regarding the color development (OD450 nm) of the labeled antibody of the room temperature storage sample, the ratio (%) where the color development (OD450 nm) of the label antibody of the −80 ° C. storage sample was 100 was compared. The results are shown in FIG.
図１１に示したＥＬＩＳＡ測定の結果から明らかなように、Ｌ−ＦＡＢＰ ＷＴは１１７〜１４３％の測定値の上昇が確認され、ＣＶは１４．５％であった。
一方、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌは１０４〜１１３％の上昇までしか認められず、ＣＶは４．８％であった。
以上の結果から３７℃での長期保存に対してＬ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌの抗体結合能は安定的であり、ＥＬＩＳＡ測定値の変動は小さいことが明らかとなった。つまり、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌは３７℃での長期保存にも安定化されたといえる。
As is clear from the results of the ELISA measurement shown in FIG. 11, the L-FABP WT was confirmed to increase in measured value by 117 to 143%, and the CV was 14.5%.
On the other hand, L-FABP M19L / M74L / M113L was observed only up to an increase of 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 and the fluctuation of the ELISA measurement value is small with respect to long-term storage at 37 ° C. That is, it can be said that L-FABP M19L / M74L / M113L was also stabilized during long-term storage at 37 ° C.
実施例５
＜クローン２とは認識部位が異なる標識抗体を用いたＥＬＩＳＡ測定における酸化安定性＞
ＡＡＰＨを所定の最終濃度（０〜４ｍＭ）になるようＬ−ＦＡＢＰ蛋白質溶液に添加し、室温で１時間反応させ、ＥＬＩＳＡ測定を実施した。標識抗体としてクローン１、クローン２、クローンＦを用い、発色（ＯＤ４５０ｎｍ）を無処理サンプルと比較した。結果を図１２に示す。
図１２に示した結果から明らかなように、クローン１及びクローンＦによる標識抗体を用いたＬ−ＦＡＢＰ測定方法では、クローン２と同様に酸化によってＬ−ＦＡＢＰ ＷＴの抗体結合能が変化すること、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌでは酸化による抗体結合能の変化が抑制されることを分かる。
したがって、抗原認識部位が異なる抗Ｌ−ＦＡＢＰ抗体クローン１、抗Ｌ−ＦＡＢＰ抗体クローンＦによるＥＬＩＳＡ測定系においても安定化されたＬ−ＦＡＢＰ蛋白質が有効である。
Example 5
<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. 12, in the L-FABP measurement method using labeled antibodies from clone 1 and clone F, the antibody-binding ability of L-FABP WT changes due to 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.
実施例６
＜脂肪酸添加による安定性＞
図１３（ａ）は、Ｌ−ＦＡＢＰ蛋白質の脂肪酸添加処理によるＥＬＩＳＡ測定値の変化（脂肪酸添加濃度０μＭを１００とした割合（％））を示す図である。
図１３（ａ）から明らかなように、結合する脂肪酸の種類（図１３（ｂ））や濃度によってＬ−ＦＡＢＰ蛋白質の抗体結合能が変化することが明らかとなった。
Example 6
<Stability by adding fatty acid>
FIG. 13 (a) is a diagram showing a change in ELISA measurement value by fatty acid addition treatment of L-FABP protein (ratio (%) where the fatty acid addition concentration is 0 μM as 100).
As is clear from FIG. 13 (a), it became clear that the antibody binding ability of the L-FABP protein varies depending on the type of fatty acid to be bound (FIG. 13 (b)) and concentration.
次に、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌに対して各種脂肪酸を最終濃度０〜７００μＭとなるよう添加し、室温で１．５時間反応させた。また空気酸化の影響を考慮し、蛋白質溶液にＤＭＳＯ（終濃度５％）のみを添加し、添加直後に測定を実施した無処理サンプルを比較対象サンプルとした。 Next, various fatty acids were added to L-FABP M19L / M74L / M113L to a final concentration of 0 to 700 μM and reacted at room temperature for 1.5 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.
これらの反応溶液及び無処理サンプルを「レナプロ Ｌ−ＦＡＢＰ テスト ＴＭＢ」を使用してＥＬＩＳＡ測定を実施し、標識抗体の発色（ＯＤ４５０ｎｍ）を無処理サンプルと比較した。上記診断用キットの使用方法は通常添付されている添付文書に従った測定方法に準じて行った。結果を図１４（ａ）〜（ｃ）に示す。 These reaction solutions and untreated samples were subjected to ELISA measurement using “Lenapro L-FABP Test TMB”, and the color development (OD450 nm) of the labeled antibody was compared with that of 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. 14 (a) to (c).
図１４（ａ）は各種濃度のアラキドン酸添加によるＥＬＩＳＡ測定値の変化を示す図であり、図１４（ｂ）は各種濃度のオレイン酸添加によるＥＬＩＳＡ測定値の変化を示す図であり、図１４（ｃ）は各種濃度の８−イソプロスタグランジンＦ２α添加によるＥＬＩＳＡ測定値の変化を示す図である。
図１４（ａ）〜（ｃ）に示したＥＬＩＳＡ測定の結果から明らかなように、Ｌ−ＦＡＢＰ ＷＴはアラキドン酸に関しては１０４〜２２０％の測定値の上昇（ＣＶ３４．０％）、オレイン酸に関しては１００〜１２４％の測定値の上昇、（ＣＶ１０．４％）、８−イソプロスタグランジンＦ２αに関しては９５〜１１８％の測定値の変動（ＣＶ６．８％）が確認された。
一方、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌはアラキドン酸に関しては８８〜１０３％の測定値の変動（ＣＶ６．１％）、オレイン酸に関しては９２〜１０７％の測定値の変動、（ＣＶ５．１％）、８−イソプロスタグランジンＦ２αに関しては９６〜１０１％の測定値の上昇（ＣＶ２．３％）が確認された。
以上の結果から脂肪酸添加に対してＬ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌの抗体結合能は安定的であり、ＥＬＩＳＡ測定値の変動は小さいことが明らかとなった。つまり、Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌは脂肪酸添加に対して安定化したといえる。
FIG. 14 (a) is a diagram showing changes in ELISA measured values by addition of various concentrations of arachidonic acid, and FIG. 14 (b) is a diagram showing changes in ELISA measured values by 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. 14 (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 above results, it was revealed that the antibody binding ability of L-FABP M19L / M74L / M113L was stable with respect to the addition of fatty acid, and the fluctuation of the ELISA measurement value was small. That is, it can be said that L-FABP M19L / M74L / M113L was stabilized against fatty acid addition.
配列番号１：Ｌ−ＦＡＢＰ ＷＴのアミノ酸配列
配列番号２：Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３Ｌのアミノ酸配列
配列番号３：Ｌ−ＦＡＢＰ Ｍ１９Ｌ／Ｍ７４Ｌ／Ｍ１１３ＬのＤＮＡ配列
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
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| JP2016120073A JP6059388B1 (en) | 2016-06-16 | 2016-06-16 | Liver-type fatty acid binding protein, DNA encoding the protein, cells transformed with the DNA, a method for producing the protein, a preparation of the protein, a method for preparing a calibration curve for the protein, and quantifying the protein Method |
| EP17813408.6A EP3421600B1 (en) | 2016-06-16 | 2017-06-15 | Liver-type fatty acid-binding protein preparation and use thereof |
| PCT/JP2017/022209 WO2017217514A1 (en) | 2016-06-16 | 2017-06-15 | Liver-type fatty acid-binding protein preparation, method for evaluating preparation, method for regulating variation range of measured value caused by liver-type fatty acid-binding protein in measurement using preparation, liver-type fatty acid-binding protein, dna encoding protein, cell transformed by dna, method for producing protein, method for drawing calibration curve for liver-type fatty acid-binding protein, and method for quantifying protein |
| US16/087,333 US10981960B2 (en) | 2016-06-16 | 2017-06-15 | Liver-type fatty acid-binding protein standard, method for evaluating standard, method for regulating variation range of measured value caused by liver-type fatty acid-binding protein in measurement using standard, liver-type fatty acid-binding protein, DNA encoding protein, cell transformed by DNA, method of producing protein, method of drawing calibration curve for liver-type fatty acid-binding protein, and method of quantifying protein |
| CN201780019737.XA CN109121419A (en) | 2016-06-16 | 2017-06-15 | Liver type fatty acid-binding protein preparation, the method that it is evaluated, the method for amplitude of fluctuation for inhibiting measured value caused by using the liver type fatty acid-binding protein in its measurement, liver type fatty acid-binding protein, its manufacturing method, encode it DNA, converted by the DNA cell, make liver type fatty acid-binding protein calibration curve method and quantitative method is carried out to the protein |
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| US10981960B2 (en) | 2016-06-16 | 2021-04-20 | Cmic Holdings Co., Ltd. | Liver-type fatty acid-binding protein standard, method for evaluating standard, method for regulating variation range of measured value caused by liver-type fatty acid-binding protein in measurement using standard, liver-type fatty acid-binding protein, DNA encoding protein, cell transformed by DNA, method of producing protein, method of drawing calibration curve for liver-type fatty acid-binding protein, and method of quantifying protein |
| CN112805565A (en) * | 2018-09-28 | 2021-05-14 | Cmic控股有限公司 | Method for quantifying liver-type fatty acid binding protein, kit for quantifying same, method for detecting renal disease, kit for detecting same, and diagnostic agent for same |
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| US10981960B2 (en) | 2016-06-16 | 2021-04-20 | Cmic Holdings Co., Ltd. | Liver-type fatty acid-binding protein standard, method for evaluating standard, method for regulating variation range of measured value caused by liver-type fatty acid-binding protein in measurement using standard, liver-type fatty acid-binding protein, DNA encoding protein, cell transformed by DNA, method of producing protein, method of drawing calibration curve for liver-type fatty acid-binding protein, and method of quantifying protein |
| JPWO2017217514A1 (en) * | 2016-12-19 | 2019-04-18 | シミックホールディングス株式会社 | 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 |
| CN112805565A (en) * | 2018-09-28 | 2021-05-14 | Cmic控股有限公司 | Method for quantifying liver-type fatty acid binding protein, kit for quantifying same, method for detecting renal disease, kit for detecting same, and diagnostic agent for same |
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