JP5706669B2 - Fusion protein, nucleic acid, vector, cell, LAB measurement method, and LAB measurement kit - Google Patents

Description

  The present invention relates to a fusion protein, a nucleic acid, a vector, a cell, a LAB (LOX-1 ligand containing ApoB) measurement method, and a LAB measurement kit, and more specifically, specifically binds to LOX-1. Fusion protein in which full-length or partial fragment of ApoB is linked to protein, nucleic acid encoding the fusion protein, vector introduced with the nucleic acid, cell containing the vector, LAB measurement method using the fusion protein, and And a LAB measurement kit containing the fusion protein.

  Low density lipoprotein (LDL) is so-called bad cholesterol and is said to be closely related to the development of arteriosclerosis. LDL is composed of apolipoprotein B (hereinafter referred to as “ApoB”) and various lipids.

  In addition, in high LDL cholesterolemia, which is an important risk factor for arteriosclerosis, it is said that oxidized LDL (oxidized LDL) bears the biological activity instead of LDL itself (non-patent literature). 1).

  As a kind of oxidized LDL receptor, a lectin-like oxidized low-density lipoprotein receptor-1 (hereinafter referred to as “LOX-1”) has been found, and its functional analysis is rapidly performed. (Non-Patent Document 1). LOX-1 is a single membrane-penetrating membrane protein whose detailed structure has already been clarified and the gene has been cloned (Patent Document 1, Non-Patent Document 1). It is also known that soluble LOX-1 (soluble LOX-1, hereinafter sometimes referred to as “sLOX-1”) exists in blood.

  Recently, a new index “LOX-1” represented by the product of the concentration of LAB (LOX-1 ligand containing ApoB) typified by oxidized LDL and the sLOX-1 concentration has been developed as a cardiovascular disorder. It has become clear that it reflects the risk of future onset of disease (CVD) (Non-Patent Document 2, Reference [4]). LAB is an in vivo substance having an activity of binding to LOX-1, and contains ApoB in its molecule. A representative example of LAB is oxidized LDL.

In order to obtain the value of the LOX-1 index, it is necessary to measure the LAB concentration and the sLOX-1 concentration in serum, and a measurement system by sandwich ELISA has been established for both. For example, LAB can be measured by sandwich ELISA using immobilized LOX-1 and anti-ApoB antibody (Non-patent Documents 1 and 2). This sandwich ELISA system strongly recognizes artificially oxidized LDL, but hardly reacts to LDL that has not been oxidized.
Thus, in the living body, there exists a lipid that binds to LOX-1 and is recognized by the anti-ApoB antibody, that is, LAB, and it can be said that artificially oxidized LDL has a property very close to that of LAB.

JP-A-9-98787

Pharmacological Journal of Japan, 119, p145-154, 2002 Clinical Chemistry, Vol.56, No.4, p550-558, 2010

  However, the present inventors have found a new problem in the process of repeatedly measuring blood LAB by the sandwich ELISA. That is, when measuring blood LAB by sandwich ELISA, a standard product (standard substance, standard protein) for drawing a calibration curve is required. The standard product conventionally used in the LAB measurement has been produced by artificially oxidizing LDL obtained from serum by ultracentrifugation in the presence of a copper ion catalyst (hereinafter referred to as “artificial oxidation LDL”). "). However, since the standard product (artificial oxidation LDL) prepared in this way is a lipid prepared from plasma, the stability is poor and long-term storage is difficult. In addition, due to its nature, it is not suitable for cryopreservation. Furthermore, it is difficult to obtain a large amount of fresh plasma as a raw material from the same person, and it is difficult to secure a large amount of artificial oxidation LDL in the same lot. In addition, it is difficult to manage the conditions of the oxidation reaction under the copper ion catalyst, and variations among lots are more likely to occur. Therefore, a researcher involved in the measurement of LAB has to measure LAB in the shortest possible time after preparation of the standard product in consideration of the stability of the standard product (artificial oxidation LDL). In addition, it is necessary to perform accuracy control by always taking into account the difference between lots of standard products.

  In view of the above-mentioned present situation, an object of the present invention is to provide a technique for measuring LAB reliably and simply, with easy accuracy control.

  The inventors of the present invention have made extensive studies on measures for solving the above problems. As a result, a fusion protein in which a protein that specifically binds to LOX-1 such as an antibody against LOX-1 (anti-LOX-1 antibody) and ApoB are fused to LOX-1 and anti-ApoB antibody It was found that the specific binding property was similar to that of artificially oxidized LDL. Furthermore, the present inventors have found that the fusion protein is excellent in stability and can be used as a new standard product that replaces the conventional artificial oxidized LDL. The present invention completed based on these new findings is as follows.

  The invention according to claim 1 is a fusion protein in which a full length or partial fragment of ApoB is linked to a protein that specifically binds to LOX-1.

  The present invention relates to a fusion protein (also referred to as a chimeric protein), wherein the full length or partial fragment of ApoB is linked to a protein that specifically binds to LOX-1. The fusion protein of the present invention has both specific binding to LOX-1 and specific binding to anti-ApoB antibody. Regarding the specific binding to LOX-1 and anti-ApoB antibody, artificial oxidized LDL and Equivalent functions are provided. For example, the fusion protein of the present invention should be used as a new standard product having excellent stability in place of the conventional standard product (artificial oxidized LDL) in LAB measurement by sandwich immunoassay using LOX-1 and anti-ApoB antibody. Can do. Moreover, mass production by recombinant DNA technology is possible, and a large amount of LAB standard products of the same lot can be provided.

  LAB (LOX-1 ligand containing ApoB) refers to an in vivo substance having an activity of binding to LOX-1 and containing ApoB in the molecule. A representative example of LAB is oxidized LDL, but it is not limited thereto as long as it has binding activity. Details of LAB are described in, for example, Kakutani M., et al., Biochem Biophys Res Commun. 2001 Mar 23; 282 (1): 180-5.

  Examples of “proteins that specifically bind to LOX-1” include antibodies to LOX-1 (anti-LOX-1 antibody), C-reactive protein (CRP), heat shock protein 70 (HSP70). ) And the like.

  The fusion protein according to claim 1, wherein the protein that specifically binds to LOX-1 is preferably an antibody against LOX-1 (claim 2).

  The invention according to claim 3 is the fusion protein according to claim 2, wherein the antibody against LOX-1 is a functional fragment of an antibody.

  In the fusion protein of the present invention, the antibody against LOX-1 is a “functional fragment of an antibody” and has a low molecular weight. Therefore, it is easy to handle and production by recombinant DNA technology is easy.

  In the fusion protein according to claim 3, the functional fragment of the antibody is preferably an Fv-type antibody (claim 4).

In the fusion protein according to any one of claims 2 to 4, the antibody against LOX-1 preferably has a configuration satisfying one or both of the following (a) and (b) for the variable region (claim 5). ).
(A) The heavy chain variable region includes a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence represented by SEQ ID NO: 3. Having a heavy chain CDR3 comprising,
(B) a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and an amino acid sequence represented by SEQ ID NO: 6 It has a light chain CDR3 containing.

  In the invention according to claim 6, the partial fragment of ApoB is at least selected from the group consisting of the region of amino acid numbers 28-97, the region of amino acid numbers 432-566, and the region of amino acid numbers 1049-1058 in human ApoB48. The fusion protein according to any one of claims 1 to 5, which comprises one region.

  Human ApoB has two isoforms, ApoB48 and ApoB100. Human ApoB48 consists of 2179 amino acids. The fusion protein of the present invention comprises an ApoB portion comprising at least one region selected from the group consisting of the region of amino acid numbers 28-97, the region of amino acid numbers 432-566, and the region of amino acid numbers 1049-1058 in human ApoB48. Has a fragment. Since the fusion protein of the present invention has a partial fragment of ApoB, the total molecular weight is small. Therefore, it is easy to handle and production by recombinant DNA technology is easy. In addition, all of the above-mentioned three regions have already been reported as epitopes of anti-ApoB antibodies, and the present inventors have newly verified the functions as epitopes this time. Therefore, the fusion protein of the present invention can reliably exhibit binding properties (reactivity) with anti-ApoB antibodies.

  The invention according to claim 7 is a nucleic acid encoding the fusion protein according to any one of claims 1 to 6.

  The invention according to claim 8 is a vector into which the nucleic acid according to claim 7 is introduced.

  The invention according to claim 9 is a cell comprising the vector according to claim 8.

  By using the nucleic acid, vector or cell of the present invention, the fusion protein of the present invention can be produced.

  The invention according to claim 10 is a method for measuring LAB using the specific binding property of LAB to LOX-1 and anti-ApoB antibody, and the fusion protein according to any one of claims 1 to 6 Is used as a standard product of LAB, and LAB in a sample is measured by comparison with the standard product.

  The present invention relates to a method for measuring LAB. The LAB measurement method of the present invention utilizes the specific binding property of LAB to LOX-1 and anti-ApoB antibody, and the fusion protein of the present invention is used as a standard product of LAB. The LAB in the sample is measured by comparing the above. That is, since the fusion protein of the present invention has both the binding ability to LOX-1 and the binding ability to the anti-ApoB antibody, it can be used as a substitute for artificial oxidation LDL. Furthermore, the fusion protein of the present invention is superior in stability compared to artificial oxidized LDL. The LAB measurement method of the present invention is easier to manage the accuracy than the conventional method using artificial oxidation LDL as a standard product, and can measure LAB reliably and easily.

  Examples of “method for measuring LAB using specific binding properties of oxidized LDL to LOX-1 and anti-ApoB antibody” include immunoassay (sandwich method, competitive method, etc.) using LOX-1 and anti-ApoB antibody ).

  The invention according to claim 11 is the method for measuring oxidized LDL according to claim 10, wherein either one of LOX-1 and anti-ApoB antibody is immobilized on a support.

  Such a configuration enables, for example, an immunoassay by a solid phase method. Examples of the support include a microtiter plate and beads.

  The invention according to claim 12 is a kit for use in the LAB measuring method according to claim 10 or 11, and the kit for measuring oxidized LDL comprising the fusion protein according to any one of claims 1 to 6. It is.

  The present invention relates to a LAB measurement kit and includes the fusion protein of the present invention. According to the LAB measurement kit of the present invention, since the fusion protein of the present invention can be used as a LAB standard, it is not necessary to prepare a LAB standard separately, which is convenient. Furthermore, since the fusion protein as the standard product is excellent in stability, the accuracy control in LAB measurement is easy, and the measurement of LAB can be performed reliably and easily.

  In the LAB measurement kit according to claim 12, a configuration further comprising LOX-1 and / or an anti-ApoB antibody is preferable (claim 13).

  The fusion protein of the present invention can be used as a new standard product having excellent stability in place of conventional artificial LDL in LAB measurement by sandwich immunoassay using LOX-1 and anti-ApoB antibody. Moreover, mass production by recombinant DNA technology is possible, and a large amount of LAB standard products of the same lot can be provided.

  By using the nucleic acid, vector, or cell of the present invention, the fusion protein of the present invention can be produced by recombinant DNA technology.

  The LAB measurement method of the present invention is easy to manage the accuracy, and can measure LAB reliably and easily.

  According to the LAB measurement kit of the present invention, the accuracy management of LAB measurement is easy, and the measurement of LAB can be performed reliably and easily.

It is explanatory drawing showing the amino acid sequence of the variable region of an anti- LOX-1 antibody, and the position of CDR1-3, (a) shows a heavy chain variable region, (b) shows a light chain variable region. (A) And (b) is explanatory drawing which shows the one aspect | mode of the connection of the LOX-1 binding protein part and ApoB part in the fusion protein of this invention. It is explanatory drawing which shows the relationship of four epitopes in human ApoB48, and ApoB fragment | piece B1-B4 containing them. It is explanatory drawing which shows the structure of the expression vector and fusion protein which were constructed | assembled in the Example. It is explanatory drawing which shows the outline | summary of the construction process of fusion protein. It is a graph which shows the result of ELISA which investigated the reactivity to the mouse | mouth anti- LOX-1 antibody to human LOX-1. It is a graph which shows the result of ELISA which investigated the reactivity to the human LOX-1 of Fv type anti- LOX-1 antibody. It is a photograph which shows the result of the western blotting with respect to the culture supernatant of a recombinant cell. It is a photograph which shows the result of the western blotting with respect to the cell lysate of a recombinant cell. It is a graph which shows the result of ELISA which investigated the reactivity with respect to the ApoB fragment of a sheep anti-ApoB polyclonal antibody. It is a photograph which shows the result of the western blotting with respect to fusion protein. It is a graph which shows the result of ELISA which investigated the reactivity with respect to human LOX-1 of a fusion protein. It is a photograph which shows the result of the western blotting which investigated the reactivity with respect to human LOX-1 of a fusion protein. It is a graph which shows the result of ELISA which investigated the reactivity with respect to the fusion protein of a sheep anti-ApoB polyclonal antibody. It is a graph which shows the result of having measured fusion protein by sandwich ELISA using human LOX-1 and a sheep anti-ApoB polyclonal antibody. It is a graph which shows the result of ELISA which compared the reactivity with respect to human LOX-1 of artificial oxidation LDL between lots. It is a graph which shows the result of ELISA which compared the reactivity with respect to human LOX-1 of a fusion protein between lots.

  The fusion protein of the present invention is obtained by linking the full length or partial fragment of ApoB to a protein that specifically binds to LOX-1. The fusion protein of the present invention comprises a part of “protein that specifically binds to LOX-1” (hereinafter sometimes referred to as “LOX-1 binding protein part”) and “full length or partial fragment of ApoB. "(Hereinafter may be referred to as" ApoB part ").

  A typical linkage mode between the LOX-1 binding protein portion and the full length or partial fragment of ApoB is linking via a peptide bond. In the following description, embodiments linked via peptide bonds will be described. In addition, as a connection mode other than a peptide bond, the thing which connected the LOX-1 binding protein part and the full length or partial fragment of ApoB by the synthetic chemical method is mentioned.

  As a protein that specifically binds to LOX-1, an antibody against LOX-1 (anti-LOX-1 antibody) is representative. That is, in a preferred embodiment, the protein that specifically binds to LOX-1 is an antibody against LOX-1. Hereinafter, the “antibody against LOX-1” part of the fusion protein of this embodiment may be referred to as “anti-LOX-1 antibody part”.

  The antibody against LOX-1 (anti-LOX-1 antibody) and the anti-LOX-1 antibody part in this embodiment will be described. In the present invention, the term “antibody” can usually be replaced with “immunoglobulin”.

  The antibody constituting the anti-LOX-1 antibody portion in the fusion protein of the present embodiment is not particularly limited as long as it is an antibody that binds to LOX-1. Here, the origin of LOX-1 is not particularly limited, and all LOX-1 such as human, mouse, bovine and the like can be targeted. The origin of the anti-LOX-1 antibody is not particularly limited, and any anti-LOX-1 antibody can be used as long as it binds to the desired LOX-1. As an example, mouse anti-LOX-1 monoclonal antibody # 10-1 (specifically described in Examples and References [1] described later) that specifically binds to human LOX-1 can be mentioned.

  The class (isotype) of the antibody constituting the anti-LOX-1 antibody portion is not particularly limited. For example, any class such as IgG, IgM, IgA, IgD, and IgE may be used. Further, the subclass of the antibody is not particularly limited, and for example, any subclass such as IgG1, IgG2, and IgG3 may be used as long as it is IgG.

In the present embodiment, “an antibody against LOX-1” includes both an antibody consisting of a full-length immunoglobulin and an “functional fragment of an antibody” containing an immunoglobulin variable region. Here, the functional fragment of an antibody refers to a part of the antibody that retains at least one action of the antibody on the antigen. Examples of functional fragments of antibodies include V _H , V _L , Fv (synonymous with scFv, V _H -V _L ), Fab, F (ab ′) ₂ , and the like. Furthermore, a conjugate of these functional fragments with the constant region can also be used as the functional fragment of the antibody in the present invention.

In a preferred embodiment, the anti-LOX-1 antibody moiety is an Fv type antibody. An Fv type antibody (scFv, V _H -V _L ) is a functional fragment of an antibody in which a heavy chain variable region (V _H ) and a light chain variable region (V _L ) are linked and has a relatively small molecular weight. Easy to handle.

By cloning the gene of the variable region of the anti-LOX-1 antibody, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody and the complementarity determining region (CDR) of each chain can be determined. . An example of the amino acid sequence and CDR of each variable region of the antibody constituting the “anti-LOX-1 antibody portion” in the fusion protein of this embodiment is shown in FIG. That is, in one embodiment of the fusion protein of the present invention, the antibody against LOX-1 (anti-LOX-1 antibody portion) satisfies one or both of the following (a) and (b) for the variable region: .
(A) The heavy chain variable region includes a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence represented by SEQ ID NO: 3. Having a heavy chain CDR3 comprising,
(B) a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and an amino acid sequence represented by SEQ ID NO: 6 It has a light chain CDR3 containing.

  In the above-described embodiment, the anti-LOX-1 antibody is employed as the “protein that specifically binds to LOX-1,” but the present invention is not limited thereto. Other examples of “protein that specifically binds to LOX-1” include C-reactive protein (CRP), HSP70, and the like. In the case of these proteins, as long as they specifically bind to LOX-1, it may be a partial fragment as well as its full length, or may be a mutation introduced with amino acid substitution or the like. Good.

  Next, the “full length or partial fragment of ApoB” (ApoB portion) in the fusion protein of the present invention will be described.

  The origin of ApoB is not particularly limited, and all ApoBs such as humans, mice and cows can be targeted.

  The ApoB part in the fusion protein of the present invention is composed of either “full length of ApoB” or “partial fragment of ApoB”. Any fragment may be used as long as it is specifically recognized by the anti-ApoB antibody. However, in terms of ease of handling, a partial fragment of ApoB having a small molecular weight is preferably used.

  The partial fragment of ApoB is not particularly limited as long as it is specifically recognized by the anti-ApoB antibody. As one example, a partial fragment containing a known epitope of ApoB can be used.

In a preferred embodiment, the ApoB portion comprises at least one region selected from the group consisting of the region of amino acid numbers 28-97, the region of amino acid numbers 432-566, and the region of amino acid numbers 1049-1058 in human ApoB48. These regions have already been reported as epitopes of human ApoB48 (references [5] and [6]), and the present inventors have verified the functions as epitopes this time. The ApoB portion may include only one of these regions, or may include two or more.
Here, the amino acid sequence corresponding to the base sequence of the gene (cDNA) of human ApoB48 is shown in SEQ ID NOs: 7 and 8. Further, the amino acid sequence of the region corresponding to amino acid numbers 28-97 is SEQ ID NO: 9, the amino acid sequence of the region corresponding to amino acid numbers 432-566 is SEQ ID NO: 10, and the amino acid sequence of the region corresponding to amino acid numbers 1049-1058 Are shown in SEQ ID NO: 11, respectively.

  ApoB in the present invention includes ApoB isoforms. For example, as described above, two isoforms of ApoB48 and ApoB100 are known for human ApoB. In the fusion protein of the present invention, both human ApoB48 and human ApoB100 can be employed as the ApoB moiety.

  ApoB in the present invention includes naturally occurring ApoB (native ApoB), as well as mutant ApoB into which mutations due to amino acid substitutions, deletions, insertions, etc. have been introduced, but which can be regarded as functionally equivalent to native ApoB. It is. That is, both the full length and partial fragment of the mutant ApoB can constitute the “ApoB portion” in the fusion protein of the present invention.

In the fusion protein of the present invention, the LOX-1 binding protein part (for example, anti-LOX-1 antibody part) and the ApoB part may be linked by the C-terminal of the LOX-1 binding protein part and the N-terminal of the ApoB part. And an embodiment in which the N-terminal of the LOX-1 binding protein portion and the C-terminus of the ApoB portion are connected (FIG. 2 (b)). Further, the LOX-1 binding protein part and the ApoB part may be indirectly linked via a sequence such as a spacer.
Furthermore, the fusion protein of the present invention may contain a protein or peptide other than the LOX-1 binding protein portion and the ApoB portion. For example, in addition to the intervening sequence such as the spacer described above, a sequence having a specific function or the like may be added to the N-terminus or C-terminus, or may be interposed between the LOX-1 binding protein portion and the ApoB portion. .

The fusion protein of the present invention can be produced, for example, by expressing a nucleic acid encoding the fusion protein. For example, DNA encoding a LOX-1 binding protein portion (for example, an anti-LOX-1 antibody portion) and DNA encoding the full length or partial fragment of ApoB are obtained. These DNAs can be easily obtained by a technique such as PCR based on known base sequence information. It may be obtained by chemical synthesis.
Next, the DNA encoding the LOX-1 binding protein portion and the DNA encoding the full length or partial fragment of ApoB are linked so that the frames coincide with each other to produce a chimeric gene (chimeric nucleic acid). The chimeric gene is a gene encoding the fusion protein of the present invention.
Next, the obtained chimeric gene is incorporated into an appropriate vector to prepare a recombinant vector (fusion protein expression vector). Further, the recombinant vector is introduced into an appropriate host cell to produce a recombinant cell (fusion protein expression cell).
Then, the recombinant cell can be cultured, and a desired fusion protein can be obtained from the culture.

  The vector into which the chimeric gene is incorporated is not particularly limited and may be appropriately selected depending on the type of host cell introduced thereafter. The host cell into which the recombinant vector is introduced is not particularly limited as long as the introduced vector functions. Examples include animal cells (COS cells, CHO cells, etc.), yeasts, bacteria (E. coli etc.), plant cells, insect cells, and the like.

  As a method for purifying the fusion protein, techniques usually used for protein purification such as centrifugation, salting out, membrane separation, and various chromatography can be applied as they are. In particular, since the fusion protein of the present invention has both binding ability to LOX-1 and binding ability to anti-ApoB antibody, affinity chromatography using a carrier on which LOX-1 or anti-ApoB antibody is immobilized can be used.

  Next, the LAB measurement method of the present invention will be described. The LAB measurement method of the present invention is a LAB measurement method that uses the specific binding property of LAB to LOX-1 and anti-ApoB antibody, and the fusion protein of the present invention described above is used as a standard product of LAB. Used to measure LAB in a sample by comparison with the standard product.

  In the LAB measurement method of the present invention, the fusion protein of the present invention is used as a standard product of LAB. That is, since the fusion protein of the present invention has both the binding ability to LOX-1 and the binding ability to anti-ApoB antibody, it can be used as a substitute for artificial oxidation LDL. Furthermore, since the fusion protein of the present invention is excellent in stability, accuracy control is easier than in the conventional method using artificial oxidized LDL as a standard product, and LAB can be measured reliably and easily.

A typical example of “a method for measuring LAB using the specific binding property of oxidized LDL to LOX-1 and anti-ApoB antibody” is a sandwich immunoassay using LOX-1 and anti-ApoB antibody. For example, LOX-1 is immobilized on a support such as a microtiter plate. Then, the measurement sample is brought into contact with the solid-phased LOX-1, and LAB in the sample is captured on LOX-1. Next, a labeled anti-ApoB antibody is bound to the captured LAB. Then, LAB in the sample can be measured using the bound anti-ApoB antibody as an index.
Examples of the label include enzymes (enzyme immunoassay, EIA, ELISA), fluorescent substances (fluorescent immunoassay, FIA), radioactive substances (radioimmunoassay, RIA), and the like. The anti-ApoB antibody may be either labeled or unlabeled. When unlabeled, a labeled secondary antibody that binds to the anti-ApoB antibody may be further used. The anti-ApoB antibody may be monoclonal or polyclonal.

  In the sandwich immunoassay, LOX-1 and anti-ApoB antibody may be used in reverse. That is, an anti-ApoB antibody may be immobilized and LOX-1 may be labeled and used.

  Examples of the support on which LOX-1 or anti-ApoB antibody is immobilized include a microtiter plate and beads.

  Another example of the “method for measuring LAB using the specific binding property of oxidized LDL to LOX-1 and anti-ApoB antibody” includes immunoassay by a competitive method.

  Purified LOX-1 can be obtained by, for example, recombinant DNA technology (Patent Document 1, Non-Patent Document 1). For example, a commercially available anti-ApoB antibody can be used as it is.

  In the LAB measurement method of the present invention, the fusion protein of the present invention is used as a standard product of LAB, and LAB in a sample is measured by comparison with the standard product. For example, serially diluted solutions of the fusion protein are prepared and used for the sandwich immunoassay and the like. Based on the value (for example, absorbance) obtained in this way, a LAB standard curve with fusion protein equivalent values can be created. LAB in a sample such as serum can be obtained as a fusion protein equivalent (relative value) by applying the value obtained when a similar sandwich immunoassay or the like is performed to the standard curve. By separately examining the correlation between artificially oxidized LDL (undegraded) immediately after preparation and the fusion protein and creating a conversion formula, LAB in the sample can also be obtained as an absolute value.

  The measurement sample used in the present invention is not particularly limited, and examples thereof include serum and plasma collected from humans.

The LAB measurement kit of the present invention is for use in the LAB measurement method of the present invention, and includes the above fusion protein. That is, according to the kit of the present invention, LAB can be easily measured using the fusion protein as a standard product.
In a preferred embodiment, it further comprises LOX-1 and / or anti-ApoB antibody. With this configuration, the above-described sandwich immunoassay and the like can be easily performed.

  Examples of the configuration of the kit of the present invention are given below. The kit may further contain a support (solid phase) such as a plate or a bead, a labeled secondary antibody, a chromogenic substrate, and the like.

[Example of kit configuration]
(A) LOX-1 (for solid phase)
(B) Anti-ApoB antibody (labeled or unlabeled)
(C) Fusion protein (LAB standard product)
(D) Dilution buffer

  As described above, a representative example of LAB is oxidized LDL. Therefore, the method for measuring LAB of the present invention includes a method for measuring oxidized LDL. Similarly, the LAB measurement kit of the present invention includes an oxidized LDL measurement kit.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

  First, the experimental method is described in the following (1) to (9).

(1) Mouse anti-LOX-1 monoclonal antibody The mouse anti-LOX-1 monoclonal antibody # 10-1 described in Reference [1] was used. Briefly, a recombinant protein corresponding to the amino acid 61-273 (61-273aa) portion of human LOX-1 (recombinant human LOX-1 protein; 61-273aa) (described in reference [2]) The spleen cells derived from Balb / c mice immunized were fused with the myeloma cell line P3U1. Positive clones were selected by ELISA to obtain # 10-1 clones.
The isotype of the antibody was determined using a mouse monoclonal antibody isotyping test kit (ABD) according to the attached instruction manual.

(2) Cloning of antibody variable region gene Total RNA was recovered from the # 10-1 antibody-producing hybridoma cloned in (1) above using 1 mL of TRIzol Reagent (Invitrogen). Synthesis of cDNA by reverse transcription reaction was performed from 1 μg of the recovered total RNA using Random hexamer (Invitrogen) and Super Script III Reverse Transcriptase (Invitrogen) according to the attached instruction manual. The antibody variable region heavy chain (V _H ) and light chain (V _L ) regions were prepared by using 0.5 μg of synthesized cDNA as a template, 25 pmol of IgG Primer set (Novagen) forward primer and 50 μL of 10 × Ex-taq buffer, Amplification was carried out using a reaction solution prepared by adding 4.0 mM of 5 mM dNTPs, 1.5 U of Ex-taq polymerase (Takara Bio Inc.) and 34.8 μL of distilled water to adjust the total volume to 50 μL. The PCR cycle was initially performed at 94 ° C. for 3 minutes, followed by 35 cycles (94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes), and the final extension was performed at 72 ° C. for 6 minutes. . The amplified DNA fragment was subcloned using TOPO TA Cloning Kit (Invitrogen), and the base sequences of _VH gene and _VL gene were determined using ABI PRISM Cycle sequencing kit (Applied Biosystems).

(3) Preparation of Fv-type anti-LOX-1 antibody Using cDNA prepared from # 10-1 antibody-producing hybridoma as a template, Fv-HF primer (SEQ ID NO: 17) and Fv-H-Linker-R primer (SEQ ID NO: 18) PCR was performed using the above primer set to amplify the V _H gene containing the # 10-1 antibody heavy chain secretion signal sequence. The Fv-H-Linker-R primer contains a part of the flexible linker sequence (Gly ₄ -Ser) ₃ at the 5 ′ end. Similarly, using the cDNA prepared from the # 10-1 antibody-producing hybridoma as a template, PCR was performed using a primer set of Linker-Fv-LF primer (SEQ ID NO: 19) and Fv-LR primer (SEQ ID NO: 20). _{The L} gene was amplified.
PCR conditions were as follows. The reaction solution was prepared by using 15 pmol of each primer set, 0.5 ng of cDNA, 5.0 μL of 10 × KOD plus buffer ver.2, 3.0 μL of 25 mM MgSO ₄ , 5.0 μL of 2 mM dNTPs, KOD plus DNA polymerase 1.0 to U (Toyobo Co., Ltd.) and 32.0 μL of distilled water were added to adjust the total amount to 50.0 μL. The PCR cycle was initially performed at 94 ° C. for 2 minutes, followed by 30 cycles (94 ° C. for 15 seconds, 60 ° C. for 30 seconds, 68 ° C. for 30 seconds), and the final extension performed at 68 ° C. for 2 minutes. .

Subsequently, each amplified fragment was ligated by overlap-extension PCR via a linker sequence inserted into a primer to prepare an Fv type antibody gene. That is, using the primer set of Fv-HF primer (SEQ ID NO: 17) and Fv-LR primer (SEQ ID NO: 20), the amplified V _H gene and _VL gene were linked by overlap-extension PCR. PCR conditions were as follows. The reaction solution used was 15 pmol of each primer set, 0.2 pmol of the amplified V _H gene and _VL gene, 5.0 μL of 10 × KOD plus buffer ver. 2, 3.0 μL of 25 mM MgSO ₄ , 2 mM The total amount was adjusted to 50.0 μL by adding 5.0 μL of dNTPs, 1.0 U of KOD plus DNA polymerase, and 32.0 μL of distilled water. The PCR cycle was initially performed at 94 ° C. for 2 minutes, followed by 30 cycles (94 ° C. for 15 seconds, 60 ° C. for 30 seconds, 68 ° C. for 45 seconds), and the final extension was performed at 68 ° C. for 2 minutes.
The amplified Fv type antibody gene was subcloned into pcDNA6.2 / V5 / GW / D-TOPO vector (Invitrogen) using pcDNA Gateway Directional TOPO Expression kit (Invitrogen). After cloning, the base sequence was confirmed using ABI PRISM Cycle sequencing kit.

Thereafter, the Fv antibody gene was excised using NotI and XbaI restriction enzyme sites present in the Multi cloning site of the pcDNA6.2 / V5 / GW / D-TOPO vector into which the Fv antibody gene was subcloned. Frame the excised Fv-type antibody gene so that V5 and 6xHistidin tag (His tag) are expressed at the C-terminal to the same restriction enzyme site in Multi cloning site of pEF6 / V5 / His vector (Invitrogen). Cloned.
Fv type antibody was expressed using FreeStyle 293 Expression system (Invitrogen). Purification of the Fv antibody was performed by TALON Metal Affinity Resins (Takara Bio Inc.).

(4) Cloning of ApoB48 gene A full-length human ApoB48 gene (GenBank accession no. NM000384; 6,537 bp; SEQ ID NO: 7) was obtained from the human liver cDNA library by PCR according to the following procedure. First, the following four primer sets for dividing the ApoB48 full-length gene into four gene fragments F1 to F4 (F1: 1-1864 bp, F2: 1802-4005 bp, F3: 3973-3207 bp, F4: 5137-6537 bp) Designed.
F1 primer set: ApoB-1-F primer (SEQ ID NO: 21) and ApoB-1864-R (SEQ ID NO: 22), F2 primer set: ApoB-1802-F primer (SEQ ID NO: 23) and ApoB-4005-R Primer (SEQ ID NO: 24), F3 primer set: ApoB-3973-F primer (SEQ ID NO: 25) and ApoB-5207-R primer (SEQ ID NO: 26), F4 primer set: ApoB-5137-F primer (SEQ ID NO: 27) and ApoB-6537-R primer (SEQ ID NO: 28).
Using the liver cDNA library of Human MTC Panel II (Clontech) as a template, PCR was performed using each primer set to obtain F1 to F4 gene fragments. PCR conditions were as follows. The reaction solution was 0.5 μg of human liver cDNA, 25 pmol of each primer set, 5.0 μL of 10 × KOD plus buffer ver.2, 3.0 μL of 25 mM MgSO ₄ , 5.0 μL of 2 mM dNTPs, and KOD plus DNA polymerase. 1.0 U and 32.0 μL of distilled water were added to adjust the total volume to 50.0 μL. The PCR cycle was initially performed at 94 ° C. for 2 minutes, followed by 35 cycles (94 ° C. for 15 seconds, 62 ° C. for 30 seconds, 68 ° C. for 90 seconds), and the final extension was performed at 68 ° C. for 2 minutes.
Next, each gene fragment was subcloned into pcDNA6.2 / V5 / GW / D-TOPO vector using pcDNA Gateway Directional TOPO Expression kit, and the nucleotide sequence was confirmed using ABI PRISM Cycle sequencing kit.

  Subsequently, using the NotI site in the pcDNA6.2 / V5 / GW / D-TOPO vector and the EcoRV site in the ApoB gene (SEQ ID NO: 7) base number 1821 (1821 bp), the F1 fragment (1-1864 bp) is 1 A -1821 bp region was excised and inserted into an F2 fragment (1802-4005 bp) treated with the same enzyme to produce a 1-4005 bp region of the ApoB gene (1-4005 bp fragment). Similarly, using the NotI site in the pcDNA6.2 / V5 / GW / D-TOPO vector and the SalI site in the ApoB gene 5161 bp, the 3973-3161 bp region was excised from the F3 fragment (3973-3207 bp) and treated with the same enzyme Was inserted into the F4 fragment (5137-6537 bp) to prepare a 3973-6537 bp region of the ApoB gene (3973-3537 bp fragment). Finally, using the NotI site in the pcDNA6.2 / V5 / GW / D-TOPO vector and the XhoI site in the ApoB gene 3995 bp, the 1-4005 bp fragment and the 3973-3537 bp fragment were ligated, and the human ApoB48 full-length gene (1 -6537 bp) An expression vector was prepared. The base sequence was confirmed again for the final product.

(5) Preparation of ApoB protein fragment In order to amplify the gene encoding ApoB protein fragments B1 to B4, the following four primer sets were designed. Each region of B1 to B4 was selected to include four sequences already reported as epitopes of anti-ApoB antibody in ApoB48 protein (full length) (FIG. 3, references [5] and [6]).
B1 gene primer set: B1-F primer (SEQ ID NO: 38) and B1-R primer (SEQ ID NO: 31), B2 gene primer set: B2-F primer (SEQ ID NO: 39) and B2-R primer (SEQ ID NO: 33) ), B3 gene primer set: B3-F primer (SEQ ID NO: 40) and B3-R primer (SEQ ID NO: 35), B4 gene primer set: B4-F primer (SEQ ID NO: 41) and B4-R primer (sequence) Number 37).
PCR was performed using each primer set using the ApoB48 full-length gene expression vector as a template, and each gene encoding B1 to B4 was amplified (FIG. 3). PCR conditions were as follows. The reaction solution was 50 ng of ApoB48 full-length gene expression vector, 25 pmol of each primer set, 5.0 μL of 10 × Ex-taq buffer, 4.0 μL of 2.5 mM dNTPs mix, 1.0 U of Ex-taq DNA polymerase, distilled water 36.0 μL was added to adjust the total volume to 50.0 μL. The PCR cycle was initially performed at 94 ° C. for 2 minutes, followed by 35 cycles (94 ° C. for 15 seconds, 62 ° C. for 30 seconds, 72 ° C. for 30 seconds), and the final extension was performed at 72 ° C. for 7 minutes.
Subsequently, the amplified gene fragments were combined in a frame so that the mouse antibody light chain secretion signal sequence was expressed on the N-terminal side and V5 and His tag were expressed on the C-terminal side, and pSecTag / FRT / V5-His-TOPO vector TA cloning in Invitrogen. After cloning, the base sequence was confirmed using ABI PRISM Cycle sequencing kit.
Expression of ApoB protein fragments B1 to B4 was performed using the FreeStyle 293 Expression system. After 96 hours of culture, the supernatant and cell lysate were collected, and the culture supernatant was purified by TALON Metal Affinity Resins to obtain ApoB protein fragments B1 to B4. B1 to B4 are regions corresponding to the following amino acid numbers in the full length of human ApoB (SEQ ID NO: 8). B1: 28-217, B2: 427-596, B3: 977-1063, B4: 1462-1552.

(6) Preparation of fusion protein of Fv-type anti-LOX-1 antibody and ApoB fragment (B1 to B4) The C-terminal side of Fv-type anti-LOX-1 antibody and the N-terminal side of ApoB fragment are linked via a linker sequence. The produced fusion proteins (4 types) were prepared by the following procedure (FIG. 4).

Using the Fv-type anti-LOX-1 antibody expression vector prepared in (2) above as a template, an Fv-HF primer (SEQ ID NO: 17) and an Fv-L-Linker-R primer containing a part of the linker sequence at the 5 ′ end ( PCR was performed using SEQ ID NO: 29) as a primer set, and the Fv-type anti-LOX-1 antibody gene was reamplified. PCR conditions were as follows. 50 ng template, 15 pmol of primer set, 5.0 μL of 10 × KOD plus buffer ver.2, 3.0 μL of 25 mM MgSO ₄ , 5.0 μL of 2 mM dNTPs, 1.0 U of KOD plus DNA polymerase, 32 of distilled water The total amount was adjusted to 50.0 μL by adding 0.0 μL. The PCR cycle was initially performed at 94 ° C. for 2 minutes, followed by 30 cycles (94 ° C. for 15 seconds, 60 ° C. for 30 seconds, 68 ° C. for 45 seconds), and the final extension was performed at 68 ° C. for 2 minutes.

In addition, PCR was performed using the ApoB48 full-length gene prepared in (5) above as a template, and using a primer set of the forward primer and reverse primer (4 pairs below) containing a part of the linker sequence at the 5 ′ end, and the ApoB48 fragment Each gene encoding B1-B4 was amplified.
Primer set for B1 gene with linker: Linker-B1-F primer (SEQ ID NO: 30) and B1-R primer (SEQ ID NO: 31), primer set for B2 gene with linker: Linker-B2-F primer (SEQ ID NO: 32) and B2-R primer (SEQ ID NO: 33), primer set for B3 gene with linker: Linker-B3-F primer (SEQ ID NO: 34) and B3-R primer (SEQ ID NO: 35), primer set for B4 gene with linker: Linker- B4-F primer (SEQ ID NO: 36) and B4-R primer (SEQ ID NO: 37).
PCR conditions were as follows. The reaction solution was 50 ng of template, 15 pmol of each primer set, 5.0 μL of 10 × KOD plus buffer ver.2, 3.0 μL of 25 mM MgSO ₄ , 5.0 μL of 2 mM dNTPs, 1.0 U of KOD plus DNA polymerase, The total amount was adjusted to 50.0 μL by adding 32.0 μL of distilled water. The PCR cycle was initially performed at 94 ° C. for 2 minutes, followed by 30 cycles (94 ° C. for 15 seconds, 60 ° C. for 30 seconds, 68 ° C. for 30 seconds), and the final extension was performed at 68 ° C. for 2 minutes.

The amplified Fv type antibody gene and each ApoB fragment gene were ligated by overlap-extension PCR. That is, Fv-HF primer (SEQ ID NO: 17) as a forward primer, B1-R primer (SEQ ID NO: 31), B2-R primer (SEQ ID NO: 33), B3-R primer (SEQ ID NO: 35) or B4 as a reverse primer Overlap-extension PCR was performed using the -R primer (SEQ ID NO: 37) and using the Fv type antibody gene and each ApoB fragment gene as a template. PCR conditions were as follows. The reaction solution was 0.2 pmol of Fv type antibody gene and each ApoB fragment gene, 15 pmol of primer set, 5.0 μL of 10 × KOD plus buffer ver.2, 3.0 μL of 25 mM MgSO ₄ , 5 mM of 2 mM dNTPs. 0.0 μL, 1.0 U of KOD plus DNA polymerase and 32.0 μL of distilled water were added to adjust the total volume to 50.0 μL. The PCR cycle started at 94 ° C. for 2 minutes, followed by 30 cycles (94 ° C. for 15 seconds, 30 seconds at 60 ° C. and 90 seconds at 68 ° C.), and the final extension was performed at 68 ° C. for 2 minutes.

Four kinds of amplified “Fv-type anti-LOX-1 antibody-ApoB fragment fusion protein” genes were subcloned into pcDNA6.2 / V5 / GW / D-TOPO vector using pcDNA Gateway Directional TOPO Expression kit. After cloning, the base sequence was confirmed using ABI PRISM Cycle sequencing kit. After that, the fusion protein gene is excised using NotI and XbaI restriction enzyme sites existing in the Multi cloning site of pcDNA6.2 / V5 / GW / D-TOPO vector, and in the Multi cloning site of pEF6 / V5 / His vector. The same restriction enzyme site was cloned in frame so that V5 and 6 × His tag were expressed at the C-terminus (fusion protein expression vector, FIG. 4).
Expression of the four fusion proteins (Fv-B1, Fv-B2, Fv-B3, Fv-B4) was performed using the FreeStyle 293 Expression system. Purification of each fusion protein was performed using TALON Metal Affinity Resins.

  The outline of the construction process of the fusion protein is summarized in FIG.

(7) Examination by ELISA IgG type, Fv type anti-LOX-1 antibody of mouse anti-LOX-1 antibody # 10-1, and four kinds of fusion proteins (Fv-B1, Fv-B2, Fv-B3, Fv-B4) ), The reactivity to LOX-1 was examined by ELISA. In addition, the reactivity of the anti-ApoB antibody to each ApoB fragment (B1 to B4) and four kinds of fusion proteins (Fv-B1, Fv-B2, Fv-B3, Fv-B4) was examined by ELISA.
Recombinant human LOX-1 (61-273aa) and BSA (negative control, Sigma) were used as anti-LOX-1 antibody and antigen for fusion protein. Further, as antigens for anti-ApoB antibodies, ApoB fragments (B1, B2), fusion proteins (Fv-B1, Fv-B2, Fv-B3, Fv-B4), ApoB protein (positive control, Sigma) and BSA (negative) Control) was used.

  30 μL of PBS containing recombinant human LOX-1 (61-273aa) or BSA was immobilized on a 384-well plate (Greiner) at 4 ° C. overnight (0.25 μg antigen / well). After washing each well twice with PBS, 50 μL of PBS containing 20% Immunoblock (DS Pharma) was added and blocked at room temperature for 1 hour.

  Each well was washed 3 times with PBS and then diluted with PBS containing 5% Immunoblock to 0.001 to 100 μg / mL to IgG type, Fv type anti-LOX-1 antibody of mouse anti-LOX-1 antibody # 10-1, or 30 μL of the four fusion proteins were added and incubated for 1 hour at room temperature.

  Each well was washed 3 times with PBS, 30 μL of secondary antibody diluted with PBS containing 5% Immunoblock was added, and incubated at room temperature for 1 hour. As a secondary antibody, a horseradish peroxidase (HRP) -labeled sheep anti-mouse IgG (1: Horseradish peroxidase (HRP) -labeled sheep anti-Mouse IgG (1: 2000); GE Healthcare Co., Ltd.) for detecting Fv-type anti-LOX-1 antibody and fusion protein (HRP-labeled mouse anti-V5 tag antibody (1: 2000); Nacalai Tesque) , Each used.

For ApoB fragment (B1, B2), fusion protein (Fv-B1, Fv-B2, Fv-B3, Fv-B4), or another plate on which ApoB protein is immobilized, after blocking and PBS washing, label with HRP Sheep anti-human ApoB polyclonal antibody (HRP-labeled sheep anti-human ApoB polyclonal antibody (Sheep polyclonal anti-ApoB antibody; binding site)) serially diluted 100-6.0 × 10 ⁸ times with PBS containing 5% Immunoblock Was added and incubated at room temperature for 1 hour.

  Each well was washed 5 times with PBS, and 3,3'5,5'-tetramethylbenzidine-containing substrate solution (TMB solution; Bio-Rad) was added to each well and allowed to react at room temperature. The reaction was stopped with 2.0 M sulfuric acid, and the absorbance at 450 nm (A: 450 nm) was measured.

(8) Examination by Western blotting The reactivity to LOX-1 of the Fv-type anti-LOX-1 antibody and four fusion proteins (Fv-B1, Fv-B2, Fv-B3, Fv-B4) was determined by Western blotting. Examined. In addition, the reactivity of the anti-ApoB antibody to each ApoB fragment (B1 to B4) and four fusion proteins (Fv-B1, Fv-B2, Fv-B3, Fv-B4) was examined by Western blotting. Recombinant human LOX-1 protein (61-273aa) and BSA (negative control) were used as anti-LOX-1 antibody and antigen for fusion protein. Further, as antigens for anti-ApoB antibodies, culture supernatants and cell lysates of ApoB fragment-expressing HEK293 cells, fusion proteins (Fv-B1, Fv-B2, Fv-B3, Fv-B4), Fv-type antibodies, and BSA (negative) Control) was used.

Each antigen is separated on a 10-20% gradient polyacrylamide gel (Wako Pure Chemical Industries, Ltd.) under non-reducing conditions and transferred to a PVDF membrane using an iBolt Dry Blotting system (Invitrogen). Went.
Subsequently, the PVDF membrane used for studying the reactivity of the fusion protein was 100% Block Ace (DS Pharma), the PVDF membrane for studying anti-ApoB antibody reactivity was 5% skim milk (Morinaga Milk Industry), 0.1. Each was blocked with PBS containing 1% Tween 20 (Nacalai Tesque) (PBS-T) at room temperature for 1 hour.

When an Fv-type anti-LOX-1 antibody or a fusion protein is used as the primary antibody, the PVDF membrane is immersed in a primary antibody solution diluted to 5 μg / mL with Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo Co., Ltd.) For 1 hour. Subsequently, it was immersed in a secondary antibody solution consisting of HRP-labeled mouse anti-V5 tag diluted 2000 times with Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo Co., Ltd.) and allowed to react at room temperature for 1 hour.
When an HRP-labeled sheep anti-human ApoB polyclonal antibody was used as the primary antibody, it was diluted 5000 times with PBS-T containing 5% skim milk and allowed to react at room temperature for 1 hour.
When the fusion protein expression confirmation antibody HRP-labeled mouse anti-V5 tag was used (positive control), it was diluted 2000 times with PBS-T containing 5% skim milk and allowed to react at room temperature for 1 hour.

  The band was detected using a chemiluminescent substrate Immobilon Western Chemiluminescent HRP Substrate (Millipore) according to the attached instruction, and the signal was developed using LAS-4000 mini (GE Healthcare).

(9) Production of LDL and artificial oxidation LDL Production of LDL and artificial oxidation LDL was performed according to the method of Reference [3]. That is, fresh plasma (d = 1.006) was obtained by centrifuging blood collected from EDTA from a healthy person at 3,000 rpm for 10 minutes. Next, KBr (Wako Pure Chemical Industries, Ltd.) was added to fresh plasma to a density of 1.019, and after ultracentrifugation at 58,000 rpm for 20 hours, the lower layer was recovered. Further, KBr was added to the collected fraction so as to have a density of 1.063, and after ultracentrifugation at 58,000 rpm for 20 hours, the upper layer was collected and dialyzed against 10,000 times of PBS to obtain LDL. It was. Oxidative modification of LDL was performed by adding 7.5 μM CuSO ₄ at a final concentration of LDL adjusted to 3 mg / mL and reacting at 37 ° C. for 16 hours, and then 10000-fold amount of LDL buffer (150 mM NaCl, 0.24 mM EDTA, pH 7. It was prepared by dialysis against 4) (artificial oxidation LDL). The degree of oxidation was determined by measuring the amount of thiobarbituric acid reaction product and the mobility of the agarose gel. Stored at 4 ° C. until use.

(10) Sandwich ELISA
Sandwich ELISA using LOX-1 and anti-ApoB antibody was performed by the method described in References [2] and [4]. First, 50 μL of PBS containing recombinant human LOX-1 (61-273aa) was immobilized on a 384-well plate (Greiner) at 4 ° C. overnight (0.25 μg / well). After washing twice with PBS, 80 μL of 3% BSA-containing HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.4) was added and blocked at room temperature for 2 hours.
After washing 3 times with PBS, 40 μL of a measurement sample (or standard) was added and incubated at room temperature for 2 hours. As a measurement sample (or standard), 4 types of fusion proteins (Fv-B1, Fv-B2, Fv-B3, Fv-B4) diluted with HEPES buffer containing 2 mM EDTA and 5% BSA, artificial oxidation LDL ( The above (prepared in (8)) or LDL (prepared in (8) above) was used.

After washing 3 times with PBS, 50 μL of HRP-labeled sheep anti-ApoB polyclonal antibody diluted 5000 times with HEPES buffer containing 2 mM EDTA and 1% BSA was added and incubated at room temperature for 1 hour.
After the antibody reaction, the plate was washed 5 times with PBS, TMB solution was added to the plate and allowed to react at room temperature. The reaction was stopped with 2M sulfuric acid and the absorbance at 450 nm was measured.

  The experimental results are described in the following (10) to (19).

(11) Reactivity of mouse anti-LOX-1 antibody # 10-1 to LOX-1 In the above (7), the results of ELISA in which recombinant human LOX-1 (61-273aa) or BSA is immobilized are shown. It is shown in FIG. In the figure, ● indicates LOX-1, and ○ indicates a case where BSA is solid-phased. Values represent the mean and SEM of three independent experiments. That is, anti-LOX-1 antibody # 10-1 showed dose-dependent reactivity with recombinant human LOX-1 at an antibody concentration ranging from 15 ng / mL to 33 μg / mL. On the other hand, in the antibody concentration range of 2 ng / mL to 100 μg / mL, no reactivity to BSA as a negative control was shown.
The isotype of mouse anti-LOX-1 antibody # 10-1 was IgG ₁ , κ.

(12) Identification of the variable region amino acid sequence and CDRs Fig. 1 (a) shows the heavy chain variable region amino acid sequence and CDRs 1-3 of the antibody # 10-1, and Fig. 1 (a) shows the light chain variable region amino acid sequence and CDRs 1-3. Each is shown in 1 (b). Furthermore, the cDNA base sequence and amino acid sequence of the heavy chain variable region of antibody # 10-1 are shown in SEQ ID NO: 13 and SEQ ID NO: 14, the amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO: 1, and the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO: 2 shows the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 3, respectively. Furthermore, the cDNA base sequence and amino acid sequence of the light chain variable region of antibody # 10-1 are shown in SEQ ID NO: 15 and SEQ ID NO: 16, the amino acid sequence of light chain CDR1 is shown in SEQ ID NO: 4, and the amino acid sequence of light chain CDR2 is shown in SEQ ID NO: 5 shows the amino acid sequence of the light chain CDR3 in SEQ ID NO: 6, respectively.

(13) Reactivity of Fv-type anti-LOX-1 antibody to LOX-1 Recombinant human LOX-1 (61-273aa) or Fv-type anti-LOX-1 antibody constructed in (3) above FIG. 7 shows the result of ELISA in which BSA was immobilized. In the figure, ● indicates LOX-1, and ○ indicates a case where BSA is solid-phased. Values represent the mean and SEM of three independent experiments. That is, the Fv-type anti-LOX-1 antibody showed reactivity to the recombinant human LOX-1 in an antibody concentration range of 46 ng / mL to 33 μg / mL in a dose-dependent manner. On the other hand, in the antibody concentration range of 2 ng / mL to 100 μg / mL, no reactivity to BSA as a negative control was shown.
Based on the above, it was considered that the Fv-type antibody can be used as a LOX-1 binding protein.

(14) Preparation of ApoB fragment As described in (4) and (5) above, cDNAs corresponding to four ApoB protein fragments (B1-B4) were cloned for the purpose of preparing anti-ApoB antibody binding protein ( FIG. 3), each fragment of B1-B4 was prepared as a recombinant protein. Each fragment was expressed in HEK293 cells, and expression of the recombinant protein in the collected culture supernatant and cell lysate was examined by Western blotting using the anti-V5 antibody (above (8)).
As a result, a band of about 35 kDa for the B1 fragment and a band of about 23 kDa for the B2 fragment were confirmed in both the culture supernatant (lower part of FIG. 8) and the cell lysate (lower part of FIG. 9). On the other hand, although the band of about 18 kDa of the B3 fragment and the band of about 18 kDa of the B4 fragment were both confirmed in the cell lysate (lower part of FIG. 9), they were not found in the culture supernatant (lower part of FIG. 8).

(15) Reactivity of anti-ApoB antibody to ApoB fragment (B1-B4) The reactivity of sheep anti-ApoB polyclonal antibody to ApoB fragment (B1-B4) was examined by Western blotting and ELISA.
As a result of Western blotting, the B2 fragment in the culture supernatant and the B2 fragment and B3 fragment in the cell lysate were reactive (FIG. 8, upper part, FIG. 9, upper part).

The result of ELISA is shown in FIG. In the figure, ● represents the B1 fragment, ▲ represents the B2 fragment, ○ represents the full length ApoB (positive control), and Δ represents BSA (negative control). Values represent the mean and SEM of three independent experiments.
When ELISA is performed using the B1 fragment and B2 fragment purified from the culture supernatant by His-tag, the sheep anti-ApoB polyclonal antibody has an antibody dilution ratio of 1.0 × 10 ^{2 to} 8.1 × 10 with respect to the B2 fragment. ^In the range of ³ times, antibody dilution ratio 1.0 × 10 ^{2 to} 9.0 × 10 ² times the B1 fragment, in the range of positive control full length ApoB, antibody dilution ratio 1.0 × 10 ^{2 to} 7 In the range of 3 × 10 ⁴ times, each showed a dose-dependent response. On the other hand, BSA as a negative control showed no reactivity in the antibody dilution ratio range of 1.0 × 10 ^{2 to} 5.9 × 10 ⁶ times.

  From the above, an ApoB protein fragment that can be used as an anti-ApoB antibody binding protein could be produced.

(16) Preparation of fusion protein of Fv-type anti-LOX-1 antibody and ApoB fragment (B1-B4) As described in (6) above, artificially oxidized LDL standard product in LOX-1 ligand measurement system (prepared from human plasma) For the purpose of obtaining an alternative product, a fusion protein of an Fv-type anti-LOX-1 antibody and an ApoB fragment (B1 to B4) was prepared (FIG. 5). The expression of the four types of fusion proteins (Fv-B1, Fv-B2, Fv-B3, Fv-B4) prepared was examined by Western blotting using an anti-V5 antibody. As a result, among the prepared fusion proteins, bands were confirmed in the vicinity of about 63 kDa for Fv-B1, about 60 kDa for Fv-B2, about 66 kDa for Fv-B3, and about 60 kDa for Fv-B5 (FIG. 11). ).

(17) Reactivity of each fusion protein to LOX-1 The Fv-type anti-LOX-1 antibody portion of four types of fusion proteins (Fv-B1, Fv-B2, Fv-B3, Fv-B4) is converted to LOX-1. In order to examine whether the binding activity was retained, ELISA and Western blotting using LOX-1 and anti-V5 antibody were performed.

The result of ELISA is shown in FIG. In the figure, ● represents Fv-B1, ▲ represents Fv-B2, ◯ represents Fv-B3, Δ represents Fv-B4, and □ represents an Fv-type anti-LOX-1 antibody (abbreviated as Fv-type antibody). Values represent the mean and SEM of three independent experiments.
That is, in Fv-B1 and Fv-B3, a dose response curve almost equivalent to that of the Fv-type anti-LOX-1 antibody was obtained in the range of the fusion protein addition amount of 45 ng / mL to 100 μg / mL. On the other hand, Fv-B2 and Fv-B4 showed reactivity in a dose-dependent manner with respect to LOX-1 at a fusion protein addition concentration ranging from 1.2 μg / mL to 100 μg / mL. The protein concentrations corresponding to the inflection points obtained were 80.9 μg / mL for Fv-B2 and 20.6 μg / mL for Fv-B4, which were approximately each compared to the Fv antibody (2.91 μg / mL). 27 times higher, about 7 times higher. The decrease in the ability of Fv-B2 and Fv-B4 to bind to LOX-1 may be due to the steric hindrance of the fused ApoB fragment, which may partially inhibit the binding of Fv-type antibody to LOX-1. It is done.
In addition, the Fv type antibody and the four fusion proteins did not show reactivity with the negative control BSA in the antibody addition concentration range of 1.6 ng / mL to 100 μg / mL.

  According to the results of Western blotting, Fv-B1 and Fv-B3 showed the same reactivity as Fv type antibody corresponding to the result of ELISA, and Fv-B2 and Fv-B4 had weaker reactivity than Fv type antibody. (FIG. 13).

(18) Reactivity of anti-ApoB antibody to each fusion protein Whether or not the anti-ApoB antibody used in this example recognizes the fusion protein in the same manner as the single ApoB fragment was examined by Western blotting and ELISA.

  As a result of Western blotting, the sheep anti-ApoB polyclonal antibody was strongly reactive to Fv-B2 and Fv-B3, weakly reactive to Fv-B1, and reactive to Fv-B4, Fv type antibody and BSA Was not shown (the upper part of FIG. 11). The lower part of FIG. 11 is a positive control.

The result of ELISA is shown in FIG. In the figure, ● indicates Fv-B1, ▲ indicates Fv-B2, ◯ indicates Fv-B3, Δ indicates Fv-B4, and □ indicates the case where BSA (negative control) is immobilized. Values represent the mean and SEM of three independent experiments.
That is, sheep anti-ApoB polyclonal antibody is 1.0 × against Fv-B1 and Fv-B2 within the range of antibody dilution ratios of 1.0 × 10 ^{2 to} 7.3 × 10 ⁴ times that of Fv-B3. In 10 ^{2 to} 8.1 × 10 ³ fold, reactivity was shown in a dose-dependent manner in the range of 1.0 × 10 ^{2 to} 2.7 × 10 ³ fold, and there was no reactivity to the negative control BSA. It was.
From the above results, it was considered that the fusion protein produced in this example could be detected by an anti-ApoB antibody.

(19) Sandwich ELISA using LOX-1 and anti-ApoB antibody for each fusion protein
The applicability of the fusion protein produced in this example as a standard protein for the LOX-1 ligand measurement system was examined by sandwich ELISA using LOX-1 and anti-ApoB antibody (above (10)).
The results are shown in FIG. In the figure, ● represents the case of Fv-B1, ▲ represents the case of Fv-B2, ○ represents the case of Fv-B3, and Δ represents the case of Fv-B4. Values represent the mean and SEM of three independent experiments.
That is, detection was possible in a dose-dependent manner at Fv-B2 addition concentrations of 1.23 μg / mL to 100 μg / mL and Fv-B3 addition concentrations of 0.14 μg / mL to 33.3 μg / mL. On the other hand, Fv-B1 was detected in a dose-dependent manner at an addition concentration of 1.23 μg / mL to 100 μg / mL, but the maximum response was about 3 times lower at OD450 than Fv-B2. Further, no reactivity was shown in the range of Fv-B4 addition concentration of 15 ng / mL to 100 μg / mL.
Since the reactivity of the anti-ApoB antibody is consistent with the results of the reactivity studies on the fusion protein by ELISA and Western blotting (FIGS. 12 and 14), the fusion protein bound to LOX-1 It could be detected specifically by the ApoB antibody, and could be used as a standard protein (standard product) of the LOX-1 ligand measurement system (LAB measurement) instead of artificially oxidized LDL.

(20) Evaluation of variation between lots (fusion protein, oxidized LDL)
For oxidized LDL artificially prepared from human plasma (artificial oxidized LDL), we used LOX-1 and anti-ApoB antibody to determine how much the standard curve changes depending on lots and how much it affects the measured value. The existing sandwich ELISA (above (10)) was used. Moreover, the usefulness of the fusion protein was verified by comparing the variation between lots in the fusion protein Fv-B3 produced in this example.

The results are shown in FIGS. The values in the figure represent the average and SEM of three independent experiments.
That is, in the detection system using a combination of recombinant LOX-1 and sheep anti-ApoB polyclonal antibody, artificial oxidized LDL (OxLDL) showed differences in reactivity to LOX-1 in four different lots as shown in FIG. It was. Based on this result, the protein concentration corresponding to the inflection point obtained by the 4-parameter logistic analysis is 38.6 μg / mL for artificial oxidation LDL lot 1, 17.7 μg / mL for lot 2, and for lot 3 It was 8.81 μg / mL and 8.34 μg / mL, and there was a difference of up to 4.6 times.
On the other hand, in the fusion protein Fv-B3, as shown in FIG. 17, the protein concentration corresponding to the inflection point obtained by the four parameter logistic analysis is 2.17 μg / mL in lot 1 and lot 2 Was 1.98 μg / mL, which was about 1.1 times the difference.

[References]
[1] Sugimoto, K., et al., LOX-1-MT1-MMP axis is crucial for RhoA and Rac1 activation induced by oxidized low-density lipoprotein in endothelial cells. Cardiovasc Res, 2009.
[2] Sato, Y., et al., Determination of LOX-1-ligand activity in mouse plasma with a chicken monoclonal antibody for ApoB. Atherosclerosis, 2008. 200 (2): p. 303-9.
[3] Sawamura, T., et al., An endothelial receptor for oxidized low-density lipoprotein. Nature, 1997. 386 (6620): p. 73-7.
[4] Inoue, N., et al., LOX Index, a Novel Predictive Biochemical Marker for Coronary Heart Disease and Stroke. Clin Chem, 2010. (Non-patent Document 2)
[5] Segrest, JP, et al., Structure of apolipoprotein B-100 in low density lipoproteins. J Lipid Res, 2001. 42 (9): p. 1346-67.
[6] Pease, RJ, et al., Use of bacterial expression cloning to localize the epitopes for a series of monoclonal antibodies against apolipoprotein B100. J Biol Chem, 1990. 265 (1): p. 553-68.

Claims (13)

  A fusion protein comprising a full-length or partial fragment of ApoB linked to a protein that specifically binds to LOX-1.   The fusion protein according to claim 1, wherein the protein that specifically binds to LOX-1 is an antibody against LOX-1.   The fusion protein according to claim 2, wherein the antibody against LOX-1 is a functional fragment of an antibody.   The fusion protein according to claim 3, wherein the functional fragment of the antibody is an Fv type antibody. The fusion protein according to any one of claims 2 to 4, wherein the antibody against LOX-1 satisfies any one or both of the following (a) and (b) for the variable region.
(A) The heavy chain variable region includes a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence represented by SEQ ID NO: 3. Having a heavy chain CDR3 comprising,
(B) a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and an amino acid sequence represented by SEQ ID NO: 6 It has a light chain CDR3 containing.   2. The partial fragment of ApoB comprises at least one region selected from the group consisting of the region of amino acid numbers 28-97, the region of amino acid numbers 432-566, and the region of amino acid numbers 1049-1058 in human ApoB48. The fusion protein according to any one of -5.   A nucleic acid encoding the fusion protein according to any one of claims 1 to 6.   A vector into which the nucleic acid according to claim 7 has been introduced.   A cell comprising the vector according to claim 8.   A method for measuring LAB using the specific binding property of LAB to LOX-1 and anti-ApoB antibody, wherein the fusion protein according to any one of claims 1 to 6 is used as a standard product of LAB, A LAB measurement method for measuring LAB in a sample by comparison with a standard product.   The method for measuring LAB according to claim 10, wherein either one of LOX-1 and anti-ApoB antibody is immobilized on a support.   A kit for use in the LAB measurement method according to claim 10 or 11, wherein the kit comprises the fusion protein according to any one of claims 1 to 6.   The LAB measurement kit according to claim 12, further comprising LOX-1 and / or an anti-ApoB antibody.