阅读说明：本技术 碱性磷酸酶融合抗体及其制造方法、以及免疫测定用试剂及免疫测定方法 (Alkaline phosphatase fusion antibody, method for producing same, reagent for immunoassay, and immunoassay method ) 是由 安保纮高 井出信幸 于 2021-05-27 设计创作，主要内容包括：本发明旨在解决因由化学修饰法的碱性磷酸酶(ALP)标记抗体的不一致性的问题。由来源于牛小肠或希瓦氏菌(Shewanella)属细菌的ALP和抗体相结合的ALP融合抗体解决上述的课题。(The present invention is intended to solve the problem of the inconsistency of an alkaline phosphatase (ALP) -labeled antibody due to a chemical modification method. The above object is achieved by an ALP fusion antibody in which ALP derived from a bacterium belonging to the genus Shewanella (Shewanella) or bovine small intestine is bound to an antibody.)

1. A method for producing an alkaline phosphatase fusion antibody, comprising:

culturing cells containing an expression vector containing a gene encoding alkaline phosphatase derived from a bacterium belonging to the genus bovine small intestine or Shewanella (Shewanella) and a gene encoding an antibody in a medium containing zinc ions, and

and (d) obtaining the alkaline phosphatase fusion antibody expressed by the cell.

2. The method of claim 1, wherein

The expression vector further contains a gene encoding a peptide linker between the gene encoding alkaline phosphatase and the gene encoding an antibody,

in the alkaline phosphatase fusion antibody, the alkaline phosphatase and the antibody are bound via the peptide linker.

3. The method of claim 1, wherein

On the expression vector, the gene encoding alkaline phosphatase and the gene encoding antibody are directly linked,

in the alkaline phosphatase fusion antibody, the alkaline phosphatase and the antibody are directly bound.

4. The method according to any one of claims 1 to 3, wherein the alkaline phosphatase contained in the alkaline phosphatase fusion antibody comprises 2 subunits.

5. The method according to any one of claims 1 to 4, wherein an antibody contained in the alkaline phosphatase fusion antibody binds to one or both of the 2 subunits.

6. The method according to any one of claims 1 to 5, wherein the antibody is at least 1 selected from the group consisting of IgG, reduced IgG, Fab ', VHH, Fd ', light chain, F (ab ')2, Fv and scFv.

7. The production process according to any one of claims 1 to 6, wherein the peptide linker comprises 1 amino acid sequence represented by any one of SEQ ID NOS 5 to 10.

8. The production method according to any one of claims 1 to 7, wherein the concentration of zinc ions in the medium is 0.01mM or more and 0.25mM or less.

9. An alkaline phosphatase fusion antibody, wherein an alkaline phosphatase derived from a bacterium belonging to the genus Shewanella (Shewanella) or bovine small intestine and an antibody are bound directly or via a peptide linker.

10. The alkaline phosphatase fusion antibody according to claim 9, wherein

The C-terminal amino acid residue of the alkaline phosphatase and the N-terminal amino acid residue of the antibody are bound directly or via a peptide linker, or

The N-terminal amino acid residue of the alkaline phosphatase and the C-terminal amino acid residue of the amino acid sequence of the antibody are bound directly or via a peptide linker.

11. The alkaline phosphatase fusion antibody according to claim 9 or 10, wherein the alkaline phosphatase contained in the alkaline phosphatase fusion antibody comprises 2 subunits.

12. The alkaline phosphatase fusion antibody according to any one of claims 9 to 11, wherein the antibody contained in the alkaline phosphatase fusion antibody binds to one or both of the 2 subunits.

13. The alkaline phosphatase fusion antibody according to any one of claims 9 to 12, wherein the antibody is at least 1 selected from the group consisting of IgG, reduced IgG, Fab ', VHH, Fd ', light chain, F (ab ')2, Fv and scFv.

14. The alkaline phosphatase fusion antibody according to any one of claims 9 to 13, wherein the peptide linker comprises 1 amino acid sequence represented by any one of SEQ ID NOS 5 to 10.

15. A reagent for immunoassay, comprising the alkaline phosphatase fusion antibody according to any one of claims 9 to 14.

16. An immunoassay method using the alkaline phosphatase fusion antibody according to any one of claims 9 to 14.

[ technical field ] A method for producing a semiconductor device

The present invention relates to an alkaline phosphatase fusion antibody and a method for producing the same. The present invention relates to a reagent for immunoassay containing an alkaline phosphatase fusion antibody. The present invention relates to an immunoassay method using an alkaline phosphatase fusion antibody.

[ background of the invention ]

Alkaline phosphatase (ALP) is an enzyme that hydrolyzes a phosphomonoester bond to generate inorganic phosphate, and is well used for labeling an antibody in immunoassays such as Enzyme Immunoassay (EIA). Labeling of an antibody with ALP has conventionally been performed by a chemical modification method in which ALP and an antibody are chemically bonded using a crosslinking agent or the like. For example, patent document 1 describes that ALP is bound to an antibody using a crosslinking agent, and the antibody is purified by a plurality of column chromatography methods to obtain a uniform ALP-labeled antibody.

[ Prior art documents ]

[ patent document ]

[ patent document 1 ] Japanese patent application laid-open No. 2001-183375

[ SUMMARY OF THE INVENTION ]

[ problem to be solved by the invention ]

In the chemical modification method, it is difficult to control the binding site of ALP and the antibody and the number of antibodies binding to ALP. Therefore, the ALP-labeled antibody obtained by the chemical modification method is heterogeneous. When such a heterogeneous ALP-labeled antibody is used in immunoassay, the measurement accuracy is affected. In order to obtain a uniform ALP-labeled antibody by the chemical modification method, as described in patent document 1, it is complicated to require purification several times.

The present invention aims to provide an ALP fusion antibody and a method for producing the same, and a reagent for immunoassay and a method for immunoassay.

[ MEANS FOR SOLVING PROBLEMS ] to solve the problems

The present inventors have found that an ALP fusion antibody using ALP derived from a bacterium belonging to the genus Shewanella (Shewanella) or bovine small intestine is suitable for immunoassay of EIA and the like, and completed the present invention.

The present invention provides a method for producing an ALP fusion antibody, comprising: culturing cells containing an expression vector containing a gene encoding ALP derived from a bacterium belonging to the genus Shewanella or bovine small intestine and a gene encoding an antibody in a medium containing zinc ions, and obtaining an ALP fusion antibody expressed by the cells.

The present invention provides an ALP fusion antibody in which ALP derived from a bacterium belonging to the genus Shewanella (Shewanella) or bovine small intestine and an antibody are bound directly or via a peptide linker. The present invention provides a reagent for immunoassay containing the above ALP fusion antibody. The present invention provides an immunoassay method using the above ALP fusion antibody.

[ Effect of the invention ]

The present invention provides an ALP fusion antibody having higher consistency and a method for producing the same.

[ brief description of the drawings ]

FIG. 1A is a schematic diagram showing an example of the structure of an expression vector.

FIG. 1B is a schematic diagram showing an example of the structure of an expression vector.

FIG. 1C is a schematic diagram showing an example of the structure of an expression vector.

Fig. 2A is a schematic diagram showing an example of the ALP fusion antibody according to the present embodiment.

Fig. 2B is a schematic diagram showing an example of the ALP fusion antibody according to the present embodiment.

Fig. 2C is a schematic diagram showing an example of the ALP fusion antibody according to the present embodiment.

Fig. 3 is a schematic diagram showing an example of the immunoassay kit according to the present embodiment.

Fig. 4 is a schematic diagram showing an example of the immunoassay kit according to the present embodiment.

Fig. 5 is a graph showing the results of antigen measurement by ELISA method using the ALP fusion antibody of example 1.

FIG. 6A is a graph showing ALP activity of ALP fusion antibodies obtained from cells cultured in media different in the conditions of addition of zinc ions.

FIG. 6B is a graph showing ALP activity of ALP fusion antibodies obtained from cells cultured in media different in the conditions of addition of zinc ions.

Fig. 6C is a graph showing ALP activity of the ALP fusion antibody obtained from cells cultured with media different in zinc ion concentration.

FIG. 7A shows the elution profile of an ALP-labeled antibody obtained by the chemical modification method after gel filtration.

FIG. 7B shows the elution profile of the ALP fusion antibody of the present embodiment after gel filtration.

FIG. 8A is a gel obtained by separating ALP-labeled antibodies obtained by the chemical modification method by SDS-PAGE.

FIG. 8B shows a gel obtained by separating the ALP fusion antibody of the present embodiment by SDS-PAGE.

Fig. 9A is a graph showing the background of immunoassay using an ALP-labeled antibody and an ALP fusion antibody according to the present embodiment.

Fig. 9B is a graph showing SN ratios in immunoassays using an ALP-labeled antibody and an ALP fusion antibody according to the present embodiment.

Fig. 10 is a graph showing the background and SN ratio of an immunoassay using an ALP-labeled antibody and an ALP fusion antibody according to the present embodiment.

FIG. 11A shows the elution profile of the ALP fusion antibody of example 3 (antibody example 1-2) after gel filtration.

FIG. 11B shows the elution profile of the ALP fusion antibody of example 3 (antibody examples 1-3) after gel filtration.

FIG. 11C shows the elution profile of the ALP fusion antibody of example 3 (antibody example 2-1) after gel filtration.

FIG. 11D shows the elution profile of the ALP fusion antibody of example 3 (antibody example 2-2) after gel filtration.

Fig. 11E is a graph showing ALP activity of the ALP fusion antibody of example 3.

Fig. 11F is a graph showing the results of antigen measurement by ELISA method using the ALP fusion antibody of example 3.

Fig. 12A is a graph showing ALP activity of the ALP fusion antibody of example 4.

FIG. 12B shows the elution profile of the ALP fusion antibody of example 4 after gel filtration.

FIG. 13A is a dissolution profile of the ALP fusion antibody (Fab of rabbit anti-PD-1 antibody) of example 5 after gel filtration.

FIG. 13B is a dissolution profile of the ALP fusion antibody of example 5 (Fab of rabbit anti-PD-L1 antibody) after gel filtration.

FIG. 13C is a dissolution profile of the ALP fusion antibody (Fab of mouse anti-VEGF antibody) of example 5 after gel filtration.

Fig. 14A is a graph showing ALP activity of the ALP fusion antibody of example 5 (Fab of rabbit anti-PD-1 antibody).

Fig. 14B is a graph showing ALP activity of the ALP fusion antibody of example 5 (Fab of rabbit anti-PD-L1 antibody).

FIG. 15A shows the elution profile of the ALP fusion antibody of example 6 after gel filtration.

FIG. 15B shows the elution profile of the ALP fusion antibody of example 6 after gel filtration.

FIG. 15C shows the elution profile of the ALP fusion antibody of example 6 after gel filtration.

Fig. 16 is a graph showing ALP activity of the ALP fusion antibody of example 6.

Fig. 17 is a graph showing the results of antigen measurement by ELISA method using the ALP fusion antibody of example 6.

FIG. 18A is a graph showing the luminescence intensity at the time of immunoassay using the ALP fusion antibody of example 6 with an automatic measuring apparatus.

FIG. 18B is a graph showing the SN ratio in the case of immunoassay using the ALP fusion antibody of example 6 with an automatic measuring apparatus.

FIG. 19 is a graph showing ALP activity of bacterium-derived ALP of Shewanell la in reference example.

[ detailed description ] embodiments

In the method for producing an ALP fusion antibody according to the present embodiment (hereinafter, also referred to as "production method"), cells including an expression vector containing a gene encoding ALP derived from a bacterium belonging to the genus bovine small intestine or Shewanel la and a gene encoding an antibody are cultured in a medium containing zinc ions.

In the production method of the present embodiment, a polypeptide in which ALP and an antibody are bonded by a peptide bond directly or via a peptide linker, that is, a polypeptide in which ALP and an antibody are integrated at the amino acid level is obtained. In contrast, in the chemical modification method described above, an antibody and ALP are separately obtained, and these proteins are bound to each other with a crosslinking agent to obtain an ALP-labeled antibody in which ALP is linked to the antibody via the crosslinking agent.

In the present embodiment, the types of bovine small intestine-derived ALP (BIAP) and Shewanella-derived ALP (S-AP) derived from bacteria are not particularly limited, and may be appropriately selected from publicly known BIAP and S-AP. In the production method of the present embodiment, since the ALP fusion antibody is produced by gene recombination, BIAP and S-AP preferably have determined amino acid sequences or nucleotide sequences encoding the amino acid sequences. The amino acid sequence or base sequence of BIAP and S-AP can also be obtained from a known database. Examples of the database include GeneBank.

Examples of BIAP include BIAP I, BIAP II, BIAP III, BIAP IV, BIAP V, BIAP VI, BIAP VII and the like (see Manes T. et al, (1998) J.biol.chem., vol.273, pp.23353-23360, specification of U.S. Pat. No. 6,406,899). Among them, BIAP II is also particularly preferable. The amino acid sequence of BIAPII and the base sequence encoding it are shown in SEQ ID NO 1 and 2, respectively. Manes t. et al, (1998) j.biol.chem., vol.273, pp.23353-23360 and U.S. patent No. 6,406,899 are incorporated herein by reference.

Examples of S-AP include S-AP derived from a strain selected from the group consisting of Shewanella sp T3-3 (see U.S. Pat. No. 9,133,446), Shewanella putreferans CN-32, Shewanella xiamenensis (Shewanella xiamenensis), Shewanella andersoniensis (Shewanella oneidensis), Shewanella frigidarium (Shewanella gaidii), Shewanella fryidamina (Shewanella Arctica sp.), Shewanella frigideridamina NCIMB400, and Shewanella livinensis (Shewanella livingnensis), shewanella (Shewanell la sp.) R106 (or M2), Shewanella (Shewanell la sp.) ALD9, Corewanella colwelchii liana, Shewanella (Shewanell la sp.) CG18, Shewanella vacucularia, Shewanella benthica (Shewanella vestigia) KT99, Shewanella benthica (Shewanella benthica) and Shewanella algidicola (Shewanella algidiscicola) bacteria ALP. The amino acid sequence of ALP derived from these bacteria is known per se, and can be obtained from a known database such as the database provided by NCBI (national Center for Biotechnology information). The amino acid sequence of ALP derived from a bacterium belonging to the genus Shewanella (Shewanella sp.) other than Shewanella T3-3 was registered in the database of NCBI with the accession numbers shown in the reference examples described later. U.S. patent No. 9,133,446 is incorporated by reference into this specification.

In the present embodiment, ALP derived from a bacterium selected from the group consisting of Shewanella sp T3-3, Shewanella putrefaction (Shewanella putrefiens) CN-32, Shewanella manshurica (Shewanella xiamenensis), Shewanella gladiata (Shewanella frigididimuralina), Shewanella livingstonensis (Shewanella livingstonensis) and Shewanella vesiculosus (Shewanella vesiculosus) is preferable. The amino acid sequence of S-AP derived from Shewanella sp T3-3 and the nucleotide sequence encoding the same are shown in SEQ ID NOS: 3 and 4, respectively.

In the present embodiment, the gene encoding BIAP or S-AP (hereinafter, also referred to as "ALP gene") may be cloned from genomic DNA of a bacterium belonging to the genus Shewanella (Shewanella) or bovine small intestine by a conventional method, or may be synthesized based on a known amino acid sequence or base sequence.

In the present specification, the term "antibody" encompasses full-length antibodies and fragments thereof. The full-length antibody may be any of IgG, IgA, IgM, IgD and IgE, preferably IgG. Examples of the antibody fragment include Fab, Fab ', F (ab ')2, Fd ', Fv, light chain, heavy chain variable region (VHH) of a heavy chain antibody, reduced igg (rgig), and single chain antibody (scFv).

In the present embodiment, the antibody is not particularly limited, and may be an antibody that recognizes any antigen. The antibody may be an antibody having a natural amino acid sequence (wild-type antibody) or an artificially produced antibody. The antibody artificially prepared is an antibody in which the amino acid sequence is artificially changed. Examples of such antibodies include antibodies in which the amino acid sequence of the Complementarity Determining Region (CDR) is changed, chimeric antibodies, humanized antibodies, bispecific antibodies, and the like. The antibody may also be an antibody derived from any animal. The animal is preferably a mammal, and examples thereof include rabbits, mice, alpacas, camels, rats, pigs, sheep, goats, cattle, horses, and humans.

In the present embodiment, the antibody preferably has a determined amino acid sequence or a nucleotide sequence encoding the same. Examples of such an antibody include an antibody in which the amino acid sequence of the antibody or the nucleotide sequence encoding the amino acid sequence is disclosed in a known database, and an antibody obtainable from a hybridoma producing the antibody. Examples of the database include GeneBank, abYsis, and IMGT. A gene encoding an antibody (hereinafter, also referred to as "antibody gene") can also be synthesized based on a known amino acid sequence or base sequence. When there is a hybridoma producing an antibody, the antibody gene can be obtained by a known method using RNA extracted from the hybridoma.

In the case of a hybridoma that does not produce an antibody, a hybridoma that produces an antibody can be prepared by a known method such as the method described in Kohler and Milstein, Nature, vol.256, p.495-497,1975, and the like. Alternatively, RNA obtained from peripheral blood or spleen of an animal such as a mouse or rabbit immunized with a predetermined antigen may be used. When RNA obtained from peripheral blood or spleen is used, cDNA can be synthesized from the RNA, and a Fab phage library can be prepared from the obtained cDNA, as described in example 1 described later. The gene encoding Fab can be obtained as an antibody gene by phage display method using the library.

In the present embodiment, a cell including an expression vector containing an ALP gene and an antibody gene is used. The expression vector is not particularly limited as long as it has a promoter that enables protein expression in a host cell and a desired gene can be inserted downstream of the promoter. The expression vector may be a plasmid vector or a viral vector. Commercially available expression vectors may also be used. The expression vector may contain genes other than the ALP gene and the antibody gene, as required. Examples of such a gene include a gene encoding a peptide linker described later, and a drug resistance gene.

In the present embodiment, the cell is not particularly limited as long as it can be used as a gene recombination expression system, and examples thereof include mammalian cells, insect cells, plant cells, yeast, and Escherichia coli. Cells containing the expression vector can be obtained by transforming or transfecting the cells with the expression vector containing the ALP gene and the antibody gene. Transformation and transfection can be carried out by a known method corresponding to the kind of the expression vector. Examples of such a method include lipofection, calcium phosphate method, and electroporation method. Commercially available transfection kits may also be used.

The ALP gene and the antibody gene are preferably incorporated into an expression vector in such a manner that a fusion protein of BIAP or S-AP and an antibody can be expressed. On the expression vector, the ALP gene may be integrated into either the upstream or downstream of the antibody gene. When the ALP gene is integrated upstream of the antibody gene, an ALP fusion antibody in which the C-terminal amino acid residue of BIAP or S-AP and the N-terminal amino acid residue of the antibody are bound directly or via a peptide linker described later is expressed. When the ALP gene is integrated downstream of the antibody gene, an ALP fusion antibody in which the N-terminal amino acid residue of BIAP or S-AP and the C-terminal amino acid residue of the antibody are bound directly or via a peptide linker described later is expressed.

When an antibody has all or a part of a heavy chain and all or a part of a light chain, like a full-length antibody or a fragment of Fab, the antibody gene includes a gene encoding all or a part of a heavy chain (hereinafter, also referred to as "heavy chain gene") and a gene encoding all or a part of a light chain (hereinafter, also referred to as "light chain gene"). The heavy chain gene and the light chain gene may be integrated into 1 expression vector so as to be expressed independently of each other, or may be integrated into 2 expression vectors individually. The ALP gene is preferably integrated upstream or downstream of either the heavy chain gene or the light chain gene. Thus, a fusion protein of ALP and all or a part of a heavy chain or a fusion protein of ALP and all or a part of a light chain is expressed.

In this embodiment, the ALP gene and the antibody gene may be directly linked to each other on the expression vector. At this time, an ALP fusion antibody in which BIAP or S-AP and an antibody are directly bound was expressed. In a further embodiment, a gene encoding a peptide linker (hereinafter, also referred to as "linker gene") may be further contained between the ALP gene and the antibody gene. At this time, an ALP fusion antibody in which BIAP or S-AP and an antibody are bound via a peptide linker is expressed. The amino acid sequence of the peptide linker is not particularly limited as long as it does not affect the antigen binding ability and ALP activity of the antibody. The length of the peptide linker is not particularly limited, and is, for example, 3 to 20 amino acid residues. In the present embodiment, the peptide linker is preferably any selected from the group consisting of GS1, GS2, GS3, EK1, EK2 and EK3 having the following amino acid sequences.

GS1：Gly-Gly-Gly-Gly-Ser(SEQ ID NO:5)

GS2：(Gly-Gly-Gly-Gly-Ser)₂(SEQ ID NO:6)

GS3：(Gly-Gly-Gly-Gly-Ser)₃(SEQ ID NO:7)

EK1：Glu-Ala-Ala-Ala-Lys(SEQ ID NO:8)

EK2：(Glu-Ala-Ala-Ala-Lys)₂(SEQ ID NO:9)

EK3：(Glu-Ala-Ala-Ala-Lys)₃(SEQ ID NO:10)

An example of the configuration of an expression vector containing an ALP gene and an antibody gene in this embodiment will be described with reference to FIGS. 1A to C. In FIGS. 1A to C, the expression vector is exemplified as a plasmid DNA, and the arrow indicates a promoter. In the figure, "Fd (HC)" represents a gene encoding Fd. Fd is the heavy chain portion of Fab. In the figure, "LC" represents a gene encoding a light chain, "linker" represents a linker gene, and "ALP subunit" represents an ALP gene. In general, when ALP as an enzyme exists as a homodimer, the "ALP subunit" as a protein refers to monomeric ALP.

FIG. 1A shows an expression vector in which a gene encoding Fd or light chain and an ALP gene are linked directly or via a gene encoding a peptide linker. FIG. 1B shows an expression vector in which a gene encoding VHH and an ALP gene are linked directly or via a gene encoding a peptide linker. FIG. 1C shows that, on each of the expression vectors shown in FIG. 1A, an expression vector further contains a gene encoding a light chain or Fd. However, the expression vector used in the present invention is not limited to these examples. For example, a gene encoding the full-length heavy chain or Fd' may be used instead of the gene encoding Fd. Alternatively, a gene encoding scFv may be used instead of the gene encoding VHH.

The expression vector of composition example 1-1 contained the gene encoding Fd, the linker gene and the ALP gene in the following order downstream of the promoter. The expression vector expresses a fusion protein in which the C-terminal amino acid residue of Fd and the N-terminal amino acid residue of ALP subunit are bound via a peptide linker. Fab containing the fusion protein and light chain was obtained as an ALP fusion antibody by transforming or transfecting cells with the expression vector constituting example 1-1 and an expression vector containing a gene encoding a light chain.

The expression vector of composition example 1-2 contained a gene encoding a light chain, a linker gene, and an ALP gene in the following order downstream of the promoter. The expression vector expresses a fusion protein in which the C-terminal amino acid residue of the light chain and the N-terminal amino acid residue of the ALP subunit are bound via a peptide linker. Fab containing the fusion protein and Fd was obtained as an ALP fusion antibody by transforming or transfecting cells with the expression vector of configuration example 1-2 and an expression vector containing the gene encoding Fd.

The expression vectors of composition examples 1 to 3 contained the Fd-encoding gene and the ALP gene in the following order downstream of the promoter. The expression vector expresses a fusion protein in which the C-terminal amino acid residue of Fd and the N-terminal amino acid residue of ALP subunit are directly bound. Fab containing the fusion protein and light chain was obtained as an ALP fusion antibody by transforming or transfecting cells with the expression vectors of configuration examples 1 to 3 and an expression vector containing a gene encoding a light chain.

The expression vectors of composition examples 1 to 4 contained a gene encoding a light chain and an ALP gene in the following order downstream of the promoter. The expression vector expresses a fusion protein in which the C-terminal amino acid residue of the light chain and the N-terminal amino acid residue of the ALP subunit are directly bound. Fab containing the fusion protein and Fd was obtained as an ALP fusion antibody by transforming or transfecting cells with the expression vectors of configuration examples 1 to 4 and an expression vector containing a gene encoding Fd.

The expression vector constituting example 2-1 contained the ALP gene, the linker gene and the gene encoding Fd in the following order downstream of the promoter. The expression vector expresses a fusion protein in which the N-terminal amino acid residue of Fd and the C-terminal amino acid residue of ALP subunit are bound via a peptide linker. Fab containing the above fusion protein and light chain was obtained as an ALP fusion antibody by transforming or transfecting cells with the expression vector constituting example 2-1 and an expression vector containing a gene encoding a light chain.

The expression vector constituting example 2-2 contained the ALP gene, the linker gene and the gene encoding the light chain in the following order downstream of the promoter. The expression vector expresses a fusion protein in which the N-terminal amino acid residue of the light chain and the C-terminal amino acid residue of the ALP subunit are bound via a peptide linker. Fab containing the above fusion protein and Fd was obtained as an ALP fusion antibody by transforming or transfecting cells with the expression vector constituting example 2-2 and an expression vector containing a gene encoding Fd.

The expression vectors of composition examples 2-3 contained the ALP gene and the gene encoding Fd in the following order downstream of the promoter. The expression vector expresses a fusion protein in which the N-terminal amino acid residue of Fd and the C-terminal amino acid residue of ALP subunit are directly bound. Fab containing the above fusion protein and light chain was obtained as an ALP fusion antibody by transforming or transfecting cells with the expression vector constituting examples 2-3 and an expression vector containing a gene encoding a light chain.

The expression vectors constituting examples 2 to 4 contained the ALP gene and the gene encoding the light chain in the following order downstream of the promoter. The expression vector expresses a fusion protein in which the N-terminal amino acid residue of the light chain and the C-terminal amino acid residue of the ALP subunit are directly bound. Fab containing the above fusion protein and Fd was obtained as an ALP fusion antibody by transforming or transfecting cells with the expression vector constituting examples 2 to 4 and an expression vector containing a gene encoding Fd.

The expression vector constituting example 3-1 contained the gene encoding VHH, the linker gene and the ALP gene in the following order downstream of the promoter. An ALP fusion antibody in which the C-terminal amino acid residue of VHH and the N-terminal amino acid residue of ALP subunit were bound via a peptide linker was obtained by transforming or transfecting into a cell using the expression vector constituting example 3-1.

The expression vector constituting example 3-2 contained the gene encoding VHH and ALP gene in the following order downstream of the promoter. An ALP fusion antibody in which the C-terminal amino acid residue of VHH and the N-terminal amino acid residue of ALP subunit were directly bound was obtained by transforming or transfecting into a cell using the expression vector constituting example 3-2.

The expression vectors constituting examples 3-3 contained the ALP gene, linker gene and VHH-encoding gene in the following order downstream of the promoter. An ALP fusion antibody in which the N-terminal amino acid residue of VHH and the C-terminal amino acid residue of ALP subunit were bound via a peptide linker was obtained by transforming or transfecting into cells using the expression vector constituting example 3-3.

The expression vectors constituting examples 3-4 contained the ALP gene and the gene encoding VHH in the following order downstream of the promoter. An ALP fusion antibody in which the N-terminal amino acid residue of VHH and the C-terminal amino acid residue of ALP subunit were directly bound was obtained by transforming or transfecting into cells using the expression vectors constituting examples 3-4.

The expression vector constituting example 4-1 was an expression vector containing 2 promoters, and contained a gene encoding Fd, a linker gene and an ALP gene in the following order downstream of one promoter, and a gene encoding a light chain downstream of the other promoter. A fusion protein comprising the C-terminal amino acid residue of Fd and the N-terminal amino acid residue of ALP subunit joined via a peptide linker and Fab of the light chain was obtained as an ALP fusion antibody by transformation or transfection into cells using the expression vector constituting example 4-1.

The expression vector constituting example 4-2 was an expression vector containing 2 promoters, and contained a gene encoding a light chain, a linker gene and an ALP gene in the following order downstream of one promoter, and a gene encoding Fd downstream of the other promoter. A fusion protein comprising a C-terminal amino acid residue of the light chain and an N-terminal amino acid residue of the ALP subunit joined via a peptide linker and a Fab of Fd was obtained as an ALP fusion antibody by transformation or transfection into cells using the expression vector constituting example 4-2.

The expression vector constituting example 4-3 was an expression vector containing 2 promoters, and the gene encoding Fd and the gene encoding ALP were contained in the following order downstream of one promoter, and the gene encoding light chain was contained downstream of the other promoter. A fusion protein containing Fd at the C-terminal amino acid residue and ALP subunit at the N-terminal amino acid residue was directly bound to each other, and Fab of the light chain was obtained as an ALP fusion antibody by transformation or transfection into cells using the expression vector of configuration examples 4-3.

The expression vectors constituting examples 4-4 were those containing 2 promoters, and the gene encoding the light chain and the ALP gene were contained in the following order downstream of one promoter, and the gene encoding the Fd gene was contained downstream of the other promoter. A fusion protein containing a direct bond between the C-terminal amino acid residue of the light chain and the N-terminal amino acid residue of the ALP subunit and Fab of Fd was obtained as an ALP fusion antibody by transformation or transfection into cells using the expression vectors of configuration examples 4 to 4.

The expression vector constituting example 5-1 was an expression vector containing 2 promoters, containing the ALP gene, linker gene and Fd-encoding gene in the following order downstream of one promoter, and containing the light chain-encoding gene downstream of the other promoter. A fusion protein comprising an N-terminal amino acid residue of Fd and a C-terminal amino acid residue of ALP subunit joined via a peptide linker and an Fab of a light chain was obtained as an ALP fusion antibody by transformation or transfection into cells using the expression vector of configuration example 5-1.

The expression vector constituting example 5-2 was an expression vector containing 2 promoters, containing the ALP gene, linker gene and gene encoding the light chain in the following order downstream of one promoter, and containing the gene encoding the Fd downstream of the other promoter. A fusion protein comprising an N-terminal amino acid residue of the light chain and a C-terminal amino acid residue of the ALP subunit joined via a peptide linker and a Fab of Fd was obtained as an ALP fusion antibody by transformation or transfection into cells using the expression vector constituting example 5-2.

The expression vector constituting example 5-3 was an expression vector containing 2 promoters, containing the ALP gene and the Fd gene in the following order downstream of one promoter, and containing the light chain-encoding gene downstream of the other promoter. A fusion protein containing Fd at its N-terminal amino acid residue and ALP subunit at its C-terminal amino acid residue was directly bound to Fab of the light chain, which was obtained as an ALP fusion antibody by transformation or transfection into cells using the expression vector of configuration examples 5-3.

The expression vectors of composition examples 5-4 were those containing 2 promoters, containing the ALP gene and the gene encoding the light chain in the following order downstream of one promoter, and the gene encoding the Fd downstream of the other promoter. A fusion protein containing a direct bond between the N-terminal amino acid residue of the light chain and the C-terminal amino acid residue of the ALP subunit and Fab of Fd was obtained as an ALP fusion antibody by transformation or transfection into cells using the expression vectors of configuration examples 5 to 4.

ALP is known as one of the zinc enzymes. In this embodiment, it is preferable that the cells including the expression vector containing the ALP gene and the antibody gene are cultured with a medium containing zinc ions. The concentration of zinc ions in the medium is preferably 0.01mM or more, more preferably 0.05mM or more, and still more preferably 0.07mM or more. Since too high a concentration of zinc ions in the medium may affect the possibility of cell culture, the concentration of zinc ions is preferably 0.25mM or less, more preferably 0.20mM or less, and still more preferably 0.15mM or less. The cells can be cultured by a known culture method according to the cells to be used. The culture medium containing zinc ions can be prepared by adding a compound or a solution thereof capable of supplying zinc ions to the culture medium of cells suitable for use. Such compounds are preferably salts of zinc and inorganic or organic acids. Examples of such salts include zinc chloride, zinc sulfate, and zinc acetate. In the present embodiment, when a salt of zinc and an inorganic or organic acid is added to the medium, the concentration of zinc ions in the medium can also be expressed as the final concentration of the salt.

As shown in experimental example 2, ALP in the resulting ALP fusion antibody did not show activity even when cultured using a medium containing zinc ions after the ALP fusion antibody was expressed in the cells. Thus, in the present embodiment, it is preferable to add zinc ions to the medium or to replace the medium with a medium containing zinc ions, after transformation or transfection into cells using an expression vector, until the ALP fusion antibody is expressed in the cells.

In the production method of the present embodiment, the ALP fusion antibody is recovered from the cells. Thus, the ALP fusion antibody of the present embodiment in which BIAP or S-AP and the antibody are bound directly or via a peptide linker was obtained. For example, cells expressing the ALP fusion antibody may be lysed in a solution containing an appropriate solubilizing agent, and the ALP fusion antibody released in the solution may be recovered. When the cells secrete the ALP fusion antibody into the medium, the culture supernatant is recovered. The free ALP fusion antibody can be recovered by a known method such as affinity chromatography. The recovered ALP fusion antibody can be purified by a known method such as gel filtration, if necessary.

In the ALP fusion antibody of the present embodiment, the C-terminal amino acid residue of ALP and the N-terminal amino acid residue of the antibody may be bound directly or via a peptide linker. Alternatively, in the ALP fusion antibody of the present embodiment, the N-terminal amino acid residue of ALP and the C-terminal amino acid residue of the amino acid sequence of the antibody may be bound directly or via a peptide linker.

As described above, ALP as an enzyme generally exists as a homodimer. Thus, in the present embodiment, ALP contained in the ALP fusion antibody may also contain 2 subunits. The 2 subunits may also form a dimer. In this case, the antibody contained in the ALP fusion antibody may bind to one or both of the 2 subunits. That is, the ALP fusion antibody of the present embodiment contains an antibody-bound ALP subunit and an antibody-unbound ALP subunit, or contains 2 antibody-bound ALP subunits. The ALP subunit to which the antibody is not bound can be obtained from an expression vector containing the ALP gene.

An example of the ALP fusion antibody according to the present embodiment will be described with reference to FIGS. 2A to C. In FIGS. 2A-C, "Fd (HC)" represents Fd, "LC" represents a light chain, "linker" represents a peptide linker, and "ALP subunit" represents monomeric ALP. In the figure, the line between Fd (HC) and LC represents the disulfide bond, and the line between the subunits of ALP represents the formation of the dimer. However, the ALP fusion antibody of the present embodiment is not limited to these examples. For example, Fd in ALP fusion antibodies can also be a full-length heavy chain or Fd'. In addition, VHH in the ALP fusion antibody may also be scFv.

In the ALP fusion antibody of antibody example 1-1, the ALP fusion Fab of 2 molecules in which the C-terminal amino acid residue of the heavy chain portion (Fd) and the N-terminal amino acid residue of the ALP subunit are bound via a peptide linker is formed into a complex from a dimer between the ALP subunits. In the ALP fusion antibody of antibody example 1-2, the ALP fusion Fab of 2 molecules in which the C-terminal amino acid residue of the light chain and the N-terminal amino acid residue of the ALP subunit are bound via a peptide linker forms a complex from a dimer between the ALP subunits.

In the ALP fusion antibodies of antibody examples 1-3, the ALP fusion Fab of 2 molecules in which the C-terminal amino acid residue of the heavy chain portion (Fd) and the N-terminal amino acid residue of the ALP subunit are directly bound forms a complex from a dimer between the ALP subunits. In the ALP fusion antibodies of antibody examples 1-4, the ALP fusion Fab of 2 molecules in which the C-terminal amino acid residue of the light chain and the N-terminal amino acid residue of the ALP subunit are directly bound, was formed into a complex from a dimer between the ALP subunits.

In the ALP fusion antibody of antibody example 2-1, the ALP fusion Fab of 2 molecules in which the N-terminal amino acid residue of the heavy chain portion (Fd) and the C-terminal amino acid residue of the ALP subunit are bound via a peptide linker is formed into a complex from a dimer between the ALP subunits. In the ALP fusion antibody of antibody example 2-2, the ALP fusion Fab of 2 molecules in which an amino acid residue at the N-terminus of the light chain and an amino acid residue at the C-terminus of the ALP subunit are bound via a peptide linker forms a complex from a dimer between the ALP subunits.

In the ALP fusion antibody of antibody example 2-3, the ALP fusion Fab of 2 molecules in which the N-terminal amino acid residue of the heavy chain portion (Fd) and the C-terminal amino acid residue of the ALP subunit are directly bound forms a complex from a dimer between the ALP subunits. In the ALP fusion antibodies of antibody examples 2-4, the ALP fusion Fab of 2 molecules in which the amino acid residue at the N-terminus of the light chain and the amino acid residue at the C-terminus of the ALP subunit are directly bound forms a complex from a dimer between the ALP subunits.

In the ALP fusion antibody of antibody example 3-1, the ALP fusion VHH of 2 molecules in which an amino acid residue at the C-terminus of VHH and an amino acid residue at the N-terminus of ALP subunit are bound via a peptide linker forms a complex from a dimer between ALP subunits. In the ALP fusion antibody of antibody example 3-2, the ALP fusion VHH of 2 molecules in which the amino acid residue at the C-terminus of VHH and the amino acid residue at the N-terminus of ALP subunit are directly bound forms a complex from a dimer between ALP subunits.

In the ALP fusion antibody of antibody example 3-3, the ALP fusion VHH of 2 molecules in which an amino acid residue at the N-terminus of VHH and an amino acid residue at the C-terminus of ALP subunit are bound via a peptide linker forms a complex from a dimer between ALP subunits. In the ALP fusion antibodies of antibody examples 3-4, the ALP fusion VHH of 2 molecules in which the amino acid residue at the N-terminus of VHH and the amino acid residue at the C-terminus of ALP subunit are directly bound forms a complex from a dimer between ALP subunits.

In the ALP fusion antibodies of antibody examples 3-5, ALP in which an amino acid residue at the C-terminus of VHH and an amino acid residue at the N-terminus of ALP subunit are bound via a peptide linker is fused to VHH and an ALP subunit to which the antibody is not bound forms a dimer between ALP subunits. Antibody ALP fusion antibody of examples 3-5 was obtained by transformation or transfection into cells using the expression vector constituting example 3-1 and an expression vector containing an ALP gene.

In the ALP fusion antibodies of antibody examples 3-6, ALP fusion VHH in which the C-terminal amino acid residue of VHH and the N-terminal amino acid residue of ALP subunit are directly bound and ALP subunit to which the antibody is not bound form a dimer between ALP subunits. Antibody ALP fusion antibody of examples 3-6 can be obtained by transformation or transfection into cells using the expression vector constituting example 3-2 and an expression vector containing an ALP gene.

In the ALP fusion antibodies of antibody examples 3-7, ALP in which an amino acid residue at the N-terminus of VHH and an amino acid residue at the C-terminus of ALP subunit are bound via a peptide linker is fused to VHH and an ALP subunit to which the antibody is not bound forms a dimer between ALP subunits. Antibody ALP fusion antibody of examples 3-7 can be obtained by transformation or transfection into cells using the expression vector constituting examples 3-3 and an expression vector containing an ALP gene.

In the ALP fusion antibodies of antibody examples 3-8, ALP fusion VHH in which the N-terminal amino acid residue of VHH and the C-terminal amino acid residue of ALP subunit are directly bound and ALP subunit to which the antibody is not bound form a dimer between ALP subunits. ALP fusion antibodies of antibody examples 3-8 can be obtained by transformation or transfection into cells using the expression vectors constituting examples 3-4 and an expression vector containing an ALP gene.

In the ALP fusion antibody of antibody example 4-1, the ALP fusion Fab in which the C-terminal amino acid residue of the heavy chain portion (Fd) and the N-terminal amino acid residue of the ALP subunit are bound via a peptide linker and the antibody-unbound ALP subunit form a dimer between the ALP subunits. ALP fusion antibody of antibody example 4-1 can be obtained by transformation or transfection into cells using, for example, the expression vector constituting example 1-1, an expression vector containing a gene encoding a light chain, and an expression vector containing an ALP gene.

In the ALP fusion antibody of antibody example 4-2, the ALP fusion Fab in which an amino acid residue at the C-terminus of the light chain and an amino acid residue at the N-terminus of the ALP subunit are bound via a peptide linker and the ALP subunit which is not bound to the antibody form a dimer between the ALP subunits. ALP fusion antibody of antibody example 4-2 can be obtained by transformation or transfection into cells using, for example, the expression vector constituting example 1-2, an expression vector containing a gene encoding Fd, and an expression vector containing an ALP gene.

In the ALP fusion antibody of antibody example 4-3, the ALP fusion Fab in which the C-terminal amino acid residue of the heavy chain portion (Fd) and the N-terminal amino acid residue of the ALP subunit are directly bound and the ALP subunit to which the antibody is not bound form a dimer between the ALP subunits. ALP fusion antibodies of antibody examples 4-3 can be obtained by transformation or transfection into cells using, for example, the expression vectors constituting examples 1-3, an expression vector containing a gene encoding a light chain, and an expression vector containing an ALP gene.

In the ALP fusion antibody of antibody example 4-4, the ALP fusion Fab in which the amino acid residue at the C-terminus of the light chain and the amino acid residue at the N-terminus of the ALP subunit are directly bound to the antibody unbound ALP subunit forms a dimer between the ALP subunits. ALP fusion antibodies of antibody examples 4-4 can be obtained by transformation or transfection into cells using, for example, the expression vectors constituting examples 1-4, an expression vector containing a gene encoding Fd, and an expression vector containing an ALP gene.

In the ALP fusion antibody of antibody example 5-1, the ALP fusion Fab in which the N-terminal amino acid residue of the heavy chain portion (Fd) and the C-terminal amino acid residue of the ALP subunit are bound via a peptide linker and the antibody-unbound ALP subunit form a dimer between the ALP subunits. Antibody ALP fusion antibody of example 5-1 can be obtained by transformation or transfection into cells using, for example, the expression vector constituting example 2-1, an expression vector containing a gene encoding a light chain, and an expression vector containing an ALP gene.

In the ALP fusion antibody of antibody example 5-2, the ALP fusion Fab in which an amino acid residue at the N-terminus of the light chain and an amino acid residue at the C-terminus of the ALP subunit are bound via a peptide linker and the ALP subunit which is not bound to the antibody form a dimer between the ALP subunits. Antibody ALP fusion antibody of example 5-2 can be obtained by transformation or transfection into cells using, for example, the expression vector constituting example 2-2, an expression vector containing a gene encoding Fd, and an expression vector containing an ALP gene.

In the ALP fusion antibody of antibody example 5-3, the ALP fusion Fab in which the N-terminal amino acid residue of the heavy chain portion (Fd) and the C-terminal amino acid residue of the ALP subunit are directly bound and the ALP subunit to which the antibody is not bound form a dimer between the ALP subunits. Antibody ALP fusion antibody of examples 5-3 can be obtained by transformation or transfection into cells using, for example, the expression vector constituting examples 2-3, an expression vector containing a gene encoding a light chain, and an expression vector containing an ALP gene.

In the ALP fusion antibody of antibody example 5-4, the ALP fusion Fab in which the amino acid residue at the N-terminus of the light chain and the amino acid residue at the C-terminus of the ALP subunit are directly bound to the antibody unbound ALP subunit forms a dimer between the ALP subunits. ALP fusion antibodies of antibody examples 5-4 can be obtained by transformation or transfection into cells using, for example, the expression vectors constituting examples 2-4, an expression vector containing a gene encoding Fd, and an expression vector containing an ALP gene.

The ALP fusion antibody of the present embodiment can be used for detecting a test substance in an immunoassay. Accordingly, one embodiment of the present invention is a reagent for immunoassay containing the ALP fusion antibody. The kind of immunoassay is not particularly limited, and can be suitably selected from known immunoassay methods such as ELISA, Western blotting, and immunocomplex transfer (see Japanese unexamined patent publication No. 1-254868). Among them, the ELISA method is also preferable. The ELISA method may be any of sandwich method, competitive binding method, direct method, indirect method, and the like.

In the present embodiment, the form of the reagent is not particularly limited, and may be a solid (for example, powder, crystal, freeze-dried product, etc.) or a liquid (for example, solution, suspension, emulsion, etc.). When the reagent is a liquid, the solvent is not particularly limited as long as it can dissolve and store the ALP fusion antibody of the present embodiment. Examples of the solvent include water, physiological saline, phosphate buffered physiological saline (PBS), Tris buffered physiological saline (TBS), and Good's buffer. Examples of Good buffers include MES, Bis-Tris, ADA, PIPES, Bis-Tris-Propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricine, Tris, Bicine, and TAPS.

The reagent for immunoassay according to the present embodiment may contain known additives. Examples of the additive include protein stabilizers such as Bovine Serum Albumin (BSA), preservatives such as sodium azide, and inorganic salts such as sodium chloride.

In the present embodiment, the container containing the reagent for immunoassay may be packaged in a box or the like and provided to the user as a kit. In the case, the file can be also attached. In the accompanying documents, the composition, the method of use, the method of storage, and the like of the reagent for immunoassay may be described. An example of the kit is shown in FIG. 3. In fig. 3, 11 denotes a kit of the present embodiment, 12 denotes a 1 st container containing an immunoassay reagent containing the ALP fusion antibody of the present embodiment, 13 denotes a packing box, and 14 denotes an attached document.

When the immunoassay is a measurement by the sandwich ELISA method, a capture substance that specifically binds to a test substance is used in addition to the ALP fusion antibody of the present embodiment. Accordingly, a further embodiment of the present invention is a kit for immunoassay comprising the 1 st reagent containing the above-described ALP fusion antibody and the 2 nd reagent containing a capture substance that specifically binds to a test substance. The details of the reagent 1 are the same as those described above for the reagent for immunoassay of the present embodiment.

The capture substance that specifically binds to the test substance is a substance for capturing the test substance on the solid phase by itself being immobilized on the solid phase. The type of the capturing substance is not particularly limited, and may be appropriately selected depending on the type of the test substance. Examples of the capture substance include an antibody, an antigen, an oligonucleotide probe, a receptor, a ligand that binds to the receptor, an aptamer, and the like. Hereinafter, the antibody used as the capture substance is also referred to as "capture antibody". The capture antibody may be any of a monoclonal antibody and a polyclonal antibody, preferably a monoclonal antibody. When the capture antibody is a monoclonal antibody, the epitope recognized by the capture antibody is preferably different from the epitope recognized by the ALP fusion antibody of the present embodiment.

In the kit for immunoassay of the present embodiment, the ALP fusion antibody of the present embodiment contained in the 1 st reagent is preferably used as a detection antibody in the sandwich ELISA method. The detection antibody is an antibody which specifically binds to a test substance and has a labeling substance, and is an antibody which provides a signal capable of detection by the labeling substance. The detection antibody is preferably not immobilized on a solid phase.

In the present embodiment, the containers each containing the 1 st reagent and the 2 nd reagent may be packaged in a box or the like and provided to the user as a reagent kit. In the case, the file can be also attached. The configuration of the immunoassay kit of the present embodiment, the composition of each reagent, the method of use, the method of storage, and the like may be described in the attached documents. An example of the kit is shown in FIG. 4. In fig. 4, 21 denotes a kit of the present embodiment, 22 denotes a 1 st container containing a 1 st reagent containing the ALP fusion antibody of the present embodiment, 23 denotes a 2 nd container containing a 2 nd reagent containing a capture substance that specifically binds to a test substance, 24 denotes a packing box, and 25 denotes an attached document.

The immunoassay kit of the present embodiment may further comprise a solid phase for immobilizing the capture substance. The solid phase may be any insoluble carrier capable of immobilizing the captured substance. The raw material of the solid phase is not particularly limited, and may be selected from, for example, organic polymer compounds, inorganic compounds, biopolymers, and the like. Examples of the organic polymer compound include latex, polystyrene, polypropylene, and the like. Examples of the inorganic compound include magnetic substances (iron oxide, chromium oxide, ferrite, and the like), silicon oxide, aluminum oxide, and glass. Examples of the biopolymer include insoluble agarose, insoluble dextran, gelatin, cellulose, and the like. Combinations of 2 or more of these may also be used. The shape of the solid phase is not particularly limited, and examples thereof include a microplate, a microtube, a test tube, a particle, and a membrane. Among them, microplates and particles (particularly magnetic particles) are also preferable.

The immunoassay kit of the present embodiment may further contain a substrate for ALP. Examples of the substrate include chemiluminescent substrates such as CDP-Star (registered trademark) (4-chloro-3- (methoxyspiro [1, 2-dioxetane-3, 2' - (5' -chloro) tricyclo [3.3.1.13,7] decan ] -4-yl) phenylphosphate 2 sodium), CSPD (registered trademark) (3- (4-methoxyspiro [1, 2-dioxetane-3, 2- (5' -chloro) tricyclo [3.3.1.13,7] decan ] -4-yl) phenylphosphate 2 sodium), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate 2 sodium, and p-nitrophenyl phosphate.

The immunoassay kit of the present embodiment may further contain a calibrator for the test substance. An example of the calibrator is a calibrator for quantifying a test substance. The calibrator may also be provided, for example, with a buffer containing no test substance (negative control) and a buffer containing a test substance at a known concentration. When the test substance is a protein, the test substance contained in the calibrator may be a recombinant protein or a polypeptide synthesized based on the amino acid sequence of the test substance.

A further embodiment of the present invention is an immunoassay method using the above ALP fusion antibody. This measurement method includes, for example, measuring a test substance in a sample using the ALP fusion antibody of the present embodiment. The test substance is not particularly limited as long as it can specifically bind to the ALP fusion antibody of the present embodiment. Examples of the test substance include proteins, nucleic acids, polysaccharides, lipids, haptens, compounds, bacteria, viruses, and the like. The sample is not particularly limited as long as it contains a test substance. Examples of the sample include biological samples such as blood and lymph, excrement such as urine and feces, and environmental samples such as river water, sea water, and soil.

The following description will discuss, as an example, the measurement by the sandwich ELISA method. In this example, the ALP fusion antibody of the present embodiment is used as a detection antibody. First, a complex containing a test substance, a capture substance that specifically binds to the test substance, and an ALP fusion antibody (detection antibody) according to the present embodiment is formed on a solid phase. The complex can be formed by mixing a sample that may contain a test substance, a capture substance, and a detection antibody. Further, the complex can be formed on a solid phase by bringing a solution containing the complex into contact with the solid phase to which a capture substance can be immobilized. Alternatively, a solid phase on which a capture substance is immobilized in advance may be used. That is, the complex can be formed on the solid phase by bringing the solid phase on which the capture substance is immobilized, the sample that can contain the test substance, and the detection antibody into contact with each other. Further, the complex formed on the solid phase can be detected by a method known in the art, whereby the test substance contained in the sample can be measured. In this embodiment, the test substance in the sample can be measured by detecting a signal generated by ALP contained in the detection antibody.

The embodiment of the immobilization onto the solid phase of the capturing substance is not particularly limited. For example, the capture substance may be directly bound to the solid phase, or the capture substance may be indirectly bound to the solid phase via another substance. Examples of the direct bonding include physical adsorption. Examples of indirect binding include binding via a combination of biotin and avidin. In this case, the capture substance is previously modified with biotin, and avidin is previously bonded to the solid phase, whereby the capture substance and the solid phase can be indirectly bonded to each other through the bonding between biotin and avidin. The biotin group includes biotin analogues such as biotin and desthiobiotin. The avidin includes avidin and avidin analogs such as streptavidin and Tamavidin (registered trademark).

In this embodiment, B/F (Bound/Free) separation may be performed between the formation of the complex and the detection of the complex, in which unreacted Free components that have not formed the complex are removed. The unreacted free component means a component that does not constitute a complex. Examples thereof include a capture substance and a detection antibody which do not bind to a test substance. The means for B/F separation is not particularly limited, and when the solid phase is a particle, only the solid phase of the captured complex is recovered by centrifugation, whereby B/F separation can be performed. When the solid phase is a vessel such as a microplate or a microtube, B/F separation can be performed by removing a solution containing unreacted free components. When the solid phase is magnetic particles, the liquid containing unreacted free components can be sucked through the nozzle in a state where the magnetic particles are magnetically bound by a magnet, whereby B/F separation can be performed, which is preferable from the viewpoint of automation. After removing unreacted free components, the solid phase of the captured complex may be washed with a suitable aqueous medium such as PBS.

In the present specification, the term "detection signal" includes the presence or absence of a qualitative detection signal, the quantification of signal intensity, and the semi-quantitative detection signal intensity. The semi-quantitative detection means that the intensity of a signal is expressed in stages as "signal does not occur", "weak", "medium", "strong", and the like. In this embodiment, the intensity of the signal is preferably quantitatively or semi-quantitatively detected.

Methods of detecting signals are known per se in the prior art. In this embodiment, the measurement can be performed by measuring signals such as light and color generated by the reaction between the complex on the solid phase and the substrate of ALP using a known device such as a spectrophotometer. The substrate for ALP is as described above.

The result of detection of the signal can be used as the result of measurement of the test substance. For example, in the case of quantifying the intensity of a signal, the measurement value itself of the intensity of the signal or a value obtained from the measurement value may be used as the measurement result of the test substance. Examples of the value obtained from the measured value of the signal intensity include a value obtained by subtracting the measured value of the negative control sample or the background value from the measured value. Alternatively, the measured value of the signal intensity may be applied to a calibration curve to determine the value of the amount or concentration of the test substance. The negative control sample can be appropriately selected, and examples thereof include samples known to contain no test substance.

In the present embodiment, it is preferable to measure the test substance in the sample by a sandwich ELISA method using the capture antibody immobilized on the magnetic particle and the ALP fusion antibody (detection antibody) of the present embodiment. In this case, the measurement can be performed using a commercially available full-automatic immunoassay apparatus such as HISCL series (Sysmex corporation).

The present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

[ examples ] A method for producing a compound

Example 1: preparation of bovine intestinal derived ALP fusion antibody

A fusion protein in which Fab of a rabbit antibody and a bovine small intestine-derived ALP subunit are combined was prepared. The fusion protein forms a dimer through ALP subunits, and becomes an ALP fusion antibody with 2-molecule Fab. This corresponds to the ALP fusion antibody having the structure shown in antibody example 1-1 described above.

(1) Preparation of expression vector

(1.1) preparation of Gene of Rabbit antibody

Lymphocytes were obtained from peripheral blood of rabbits immunized with CD80, and mRNA was extracted from the lymphocytes to synthesize cDNA. The obtained cDNA was amplified using known primers for cloning antibody genes, and a Fab phage library was prepared. Using the resulting library, a clone of rabbit anti-CD 80 Antibody was obtained by well-known Fab Phage Display method and biopanning (see Lang IM, Barbas CF 3rd, Schleef RR., Recombinant rabbit Fab with binding activity to type-1 plasmid activator inhibitor derivative from a phase-Display library of human alpha-grams, (1996) Gene 172(2):295-8 and Philippia M.O' Brien, Robert Aitken, Antibody phase Display, (2002) Methods in Molecular Biology Volume No. 178). The gene of the obtained Fab clone of the rabbit anti-CD 80 antibody was integrated with a plasmid DNA containing a gene encoding the Fc region of the rabbit antibody, and a plasmid DNA containing the gene of the rabbit anti-CD 80 antibody was obtained. The amino acid sequences of Fd (heavy chain portion of Fab) and light chain of the rabbit anti-CD 80 antibody are shown in SEQ ID NOS: 11 and 12, respectively. Further, the nucleotide sequences encoding the amino acid sequences are shown in SEQ ID NOS 13 and 14, respectively.

(1.2) preparation of Gene derived from bovine intestinal ALP

A plasmid DNA containing the BIAPII gene was obtained by gene synthesis with GenScript having the amino acid sequence of BIAPII described in U.S. Pat. No. 6,406,899. The amino acid sequence of BIAPII and the base sequence encoding it are shown in SEQ ID NO 1 and 2, respectively.

(1.3) preparation of Fd-linker-BIAPII expression vector

(i) Preparation of BIAPII expression vector

A DNA fragment encoding BIAPII (BIAPII insert) was obtained by PCR using plasmid DNA containing the BIAPII gene as a template. The pcDNA3.4 vector was used as a template to obtain a linearized vector DNA by the inverse PCR method. The PCR was carried out using KOD Plus neo (Toyo Boseki Co., Ltd.) according to the attached document. The base sequences of the primers used in PCR are as follows.

Primer set for vector

Forward direction: 5'-TGATAAAAGG GTTCGATCCC TACC-3' (SEQ ID NO:15)

And (3) reversing: 5'-GCAGTGCACG GTGGCGCAGT ACACC-3' (SEQ ID NO:16)

Primer set for BIAPII insert

Forward direction: 5'-GCCACCGTGC ACTGCTTAAT TCCGGCAGAA GAAGAAAACC-3' (SEQ ID NO:17)

And (3) reversing: 5'-CGAACCCTTT TATCACGCAG GTGCAGGCAA GTTACAATC-3' (SEQ ID NO:18)

A BIAPII insert was ligated to the linearized vector DNA of pcDNA3.4 using In-Fusion (registered trademark) HD Cloning Kit (Takara Shuzo Co., Ltd.) to obtain a BIAPII expression vector. The In-Fusion reaction was carried out according to the attached document of the kit.

(ii) Insertion of Fd Gene into BIAPII expression vector

A DNA fragment (Fd-linker insert) encoding Fd and a peptide linker was obtained by PCR using plasmid DNA containing the rabbit anti-CD 80 antibody gene as a template. In the resulting Fd insert, a gene encoding a peptide linker was ligated downstream of the gene encoding Fd of the rabbit anti-CD 80 antibody. The types of peptide linkers were GS1, GS2, GS3, EK1, EK2 and EK3 described above. The BIAPII expression vector is used as a template, and the linearized vector DNA containing the BIAPII gene is obtained by an inverse PCR method. The PCR was carried out using KOD Plus neo (Toyo Boseki Co., Ltd.) according to the attached document. The base sequences of the primers used in PCR are as follows.

Primer set for vector

Forward GS 1: 5'-GGTGGCGGTG GATCCTTAAT TCCGGCAGAA GAAGAAAACC-3' (SEQ ID NO:19)

Forward GS 2: 5'-GTGGATCCGG AGGGGGCGGA AGTTTAATTC CGGCAGAAGA AGAAAACC-3' (SEQ ID NO:20)

Forward GS 3: 5'-GGAGGGGGCG GAAGTGGCGG GGGAGGTTCA TTAATTCCGG CAGAAGAAGA AAACC-3' (SEQ ID NO:21)

Forward EK 1: 5'-GAAGCCGCTG CTAAGTTAAT TCCGGCAGAA GAAGAAAACC-3' (SEQ ID NO:22)

Forward EK 2: 5'-CTGCTAAGGA GGCAGCCGCG AAATTAATTC CGGCAGAAGA AGAAAACC-3' (SEQ ID NO:23)

Forward EK 3: 5'-GAGGCAGCCG CGAAAGAAGC AGCGGCTAAA TTAATTCCGG CAGAAGAAGA AAACC-3' (SEQ ID NO:24)

And (3) reversing: 5'-GCAGTGCACG GTGGCGCAGT ACACC-3' (SEQ ID NO:25)

Primer set for Fd insert

Forward direction: 5'-GCCACCGTGC ACTGCCAGTC GGTGGAGGAG TCCGG-3' (SEQ ID NO:26)

Reverse GS 1: 5'-GGATCCACCG CCACCCGTGG GCTTGCTGCA TGTCGAGGG-3' (SEQ ID NO:27)

Reverse GS 2: 5'-CCCCCTCCGG ATCCACCGCC ACCCGTGGGC TTGCTGCATG TCGAGGG-3' (SEQ ID NO:28)

Reverse GS 3: 5'-ACTTCCGCCC CCTCCGGATC CACCGCCACC CGTGGGCTTG CTGCATGTCG AGGG-3' (SEQ ID NO:29)

Reverse EK 1: 5'-CTTAGCAGCG GCTTCCGTGG GCTTGCTGCA TGTCGAGGG-3' (SEQ ID NO:30)

Reverse EK 2: 5'-GCTGCCTCCT TAGCAGCGGC TTCCGTGGGC TTGCTGCATG TCGAGGG-3' (SEQ ID NO:31)

Reverse EK 3: 5'-TTTCGCGGCT GCCTCCTTAG CAGCGGCTTC CGTGGGCTTG CTGCATGTCG AGGG-3' (SEQ ID NO:32)

An Fd insert was ligated to a linearized vector DNA containing the BIAPII gene using In-Fusion (registered trademark) HD Cloning Kit (Takara Shuzo Co., Ltd.) to obtain an Fd-linker-BIAPII expression vector. The In-Fusion reaction was carried out according to the attached document of the kit. In this expression vector, a gene encoding a peptide linker and a BIAPII gene were ligated downstream of the gene encoding Fd of the rabbit anti-CD 80 antibody. These expression vectors correspond to the expression vectors described above which constitute the structure shown in configuration example 1-1.

(1.4) preparation of light chain expression vector

A DNA fragment (LC insert) encoding the light chain was obtained by PCR using plasmid DNA containing the above-mentioned rabbit anti-CD 80 antibody gene as a template. The pcDNA3.4 vector was used as a template to obtain a linearized vector DNA by the inverse PCR method. The PCR was carried out using KOD Plus neo (Toyo Boseki Co., Ltd.) according to the attached document. The base sequences of the primers used in PCR are as follows.

Primer set for vector

Forward direction: 5'-TAATCTAGAT AATTAAAGGG TTCG-3' (SEQ ID NO:33)

And (3) reversing: 5'-GCTGCGATAG CCCGGAAACA GTACC-3' (SEQ ID NO:34)

Primer set for LC insert

Forward direction: 5'-CCGGGCTATC GCAGCGAGCT CGTGATGACC CAGAC-3' (SEQ ID NO:35)

And (3) reversing: 5'-TAATTATCTA GATTATCAAC AGTCACCCCT ATTGAAGC-3' (SEQ ID NO:36)

An LC insert was ligated to the linearized vector DNA of pcDNA3.4 using In-Fusion (registered trademark) HD Cloning Kit (Takara Shuzo Co., Ltd.) to obtain a light chain expression vector. The In-Fusion reaction was carried out according to the attached document of the kit.

(2) Production and culture of antibody-producing cells

(2.1) transfection into host cells

5% CO in a medium to which a solution of zinc chloride (Nitian chemical) was added to a final concentration of 0.1mM₂Expi293F (trade mark) cells were cultured with shaking (125rpm) at 37 ℃ under an atmosphere. 25.5mL of cell culture (3.0X 10) was prepared in a number corresponding to the number of samples⁶Individual cells/mL). An appropriate amount of Opti-MEMI (trade Mark) was added to a mixture of Fd-linker-BIAPII expression vector (about 15. mu.g) and light chain expression vector (about 15. mu.g) to prepare 1.5mL, and the mixture was stirred to prepare a DNA solution. The transfection reagent was prepared by stirring Expifactamine 293 reagent (80. mu.L) and Opti-MEMI (1.5mL) stably and allowing the mixture to stand at room temperature for 5 minutes. The DNA solution and transfection reagent were stirred steadily and allowed to stand at room temperature for 20 minutes. The resulting mixture (3mL) was added to a cell culture (25.5mL) with 5% CO₂The culture was performed under an atmosphere at 37 ℃ for 20 hours (125rpm) with shaking. After 20 hours, Expifactamine (trade Mark) transfection enhancers 1 and 2, 150. mu.L and 1.5mL, respectively, 5% CO were added to each culture₂The culture was carried out under shaking at 37 ℃ for 5 days (125rpm) under an atmosphere.

(2.2) recovery and purification of antibody

The culture supernatant was recovered 5 days after the transfection. The culture supernatant contained each ALP fusion antibody secreted from transfected Expi293F (trademark) cells. The collected culture supernatant was transferred to a centrifugal sedimentation tube and centrifuged (1000g, 5 minutes, 4 ℃ C.), and the supernatant was transferred to a new centrifugal sedimentation tube. The supernatant was centrifuged again (10000g, 10 min, 4 ℃ C.) and the supernatant was transferred to a new centrifugal settler tube. The supernatant was concentrated using Amicon Ultra-4,30k (Merck). The concentrated supernatant was purified by gel filtration using AKTA avant25(GE Healthcare) and Superdex 200Increase 10/300GL (GE Healthcare). In gel filtration, the injection amount was 500. mu.L, and a buffer for ALP (100mM Triethanolamine (TEA), 150mM NaCl, 1mM MgCl) was used in the electrophoresis buffer₂、0.1mM ZnCl₂pH 7.0) was added to the reaction mixture, and the mixture was separated at a flow rate of 1 mL/min. The concentration of gel filtration-purified ALP fusion antibody was measured from the absorbance at 280nm using nanodrop-1000(Thermo Fisher Co.).

[ test example 1: antigen detection ability of ALP fusion antibody

The antigen detection ability was examined by performing ELISA using the ALP fusion antibody of example 1 as a detection antibody.

(1) Determination by chemiluminescence ELISA

The rabbit anti-CD 80 antibody (IgG) was diluted to 1. mu.g/mL with PBS. The obtained antibody solution was added to a 96-well blackboard at 100. mu.L/well, and left to stand at 4 ℃ overnight to immobilize the antibody to the well. After removing the antibody solution from the plate, BufferI' (100mM HEPES, 1% BSA, 0.05% ProClin (trade Mark), 1mM MgCl was added at 300. mu.L/well₂、0.1mM ZnCl₂pH 8.0), closed by standing overnight at 4 ℃. After removing the solution from the plate, as an antigen solution, a human CD80/B7-1 protein (His-tag) solution (0.005, 0.020, 0.078, 0.313, 1.25, 5.0, or 20ng/mL) was added at 100 μ L/well. The plate was shaken at 500rpm for 1 hour at room temperature to carry out the antigen-antibody reaction. After washing the plate 3 times with HISCL wash solution (Sysmex corporation), each ALP fusion antibody of example 1 (200ng/mL) diluted with BufferI' was added at 100. mu.L/well. The plate was shaken at 500rpm for 1 hour at room temperature to carry out the antigen-antibody reaction. After washing the plate 3 times with HISCL rinse, 1To 00. mu.L/well of the sample was added HISCL R5 reagent (Sysmex corporation) as a solution of a chemiluminescent substrate for ALP, and the luminescence intensity was immediately measured using FLUOstar OPTIMA (BMG LABTECH).

(2) Results

The measurement results are shown in FIG. 5. In the figure, GS1, GS2, GS3, EK1, EK2 and EK3 are types of peptide linkers in the ALP fusion antibody. As shown, even if any ALP fusion antibody is used, the light emission intensity increases corresponding to the antigen concentration. Thus, it was shown that any ALP fusion antibody retained the same degree of antigen detection ability.

[ test example 2: examination of influence of Zinc ion

Cells containing the expression vector obtained in example 1 were cultured under various conditions in a zinc-supplemented medium or a zinc-unsupplemented medium, and ALP activity of the ALP fusion antibody was measured.

(1) Culture of antibody-producing cells and recovery of antibodies

(1.1) cell culture Using Zinc-free Medium

The Fd-linker-biapiii expression vector and the light chain expression vector were transfected into the cells in the same manner as in example 1, except that Expi293F (trademark) cells cultured in a normal medium to which zinc was not added were used. After 20 hours, the medium was replaced with a medium supplemented with no zinc addition or with a zinc chloride solution (final concentration 0.1 mM). Further, Expifactamine (trade Mark) transfection enhancer 1 and 2, 5% CO were added₂The culture was carried out under shaking at 37 ℃ for 5 days (125rpm) under an atmosphere. After the culture, an ALP fusion antibody (GS1 linker) was recovered and purified from the culture supernatant in the same manner as in example 1.

(1.2) cell culture Using Zinc-supplemented Medium after expression of ALP fusion antibody

Transfection, medium replacement after 20 hours, and culture for 5 days were carried out in the same manner as in (1.1) above. After 5 days, the medium was replaced with a medium to which no zinc had been added or a zinc chloride solution (final concentration: 0.1mM), and the culture was continued for another 1 hour. After the culture, the ALP fusion antibody was recovered and purified from the culture supernatant in the same manner as in example 1.

(1.3) cell culture Using high-concentration Zinc-supplemented Medium

Transfection, medium replacement after 20 hours, and culture for 5 days were carried out in the same manner as in example 1 except that the medium containing zinc chloride at a concentration of 0.25mM or 0.5mM was used. After the culture, an ALP fusion antibody (GS1 linker) was recovered and purified from the culture supernatant in the same manner as in example 1. For comparison, the ALP fusion antibody (GS1 linker) was recovered and purified in the same manner as in example 1 using a medium in which zinc chloride was present at a concentration of 0.1 mM.

(2) Measurement of ALP Activity

The ALP fusion antibody was diluted with ALP buffer. The resulting antibody solution was added to a 96-well blackboard at 10. mu.L/well. HISCL R5 reagent (Sysmex Co., Ltd.) was added at a volume of 100. mu.L/well, and the luminescence intensity was immediately measured by FLUOstar OPTIMA (BMG Labtech Co.). The measurement results are shown in FIGS. 6A to C. FIGS. 6A and B show the ALP activity of the ALP fusion antibody obtained from cells cultured with a zinc supplemented medium after transfection, and cells cultured with a zinc supplemented medium after expression of the ALP fusion antibody, assuming that the ALP activity of the ALP fusion antibody obtained from cells cultured with a zinc supplemented medium after transfection is 100%. FIG. 6C shows ALP activity of ALP fusion antibody obtained from cells cultured with a medium having a concentration of zinc chloride of 0.25mM or 0.5mM, assuming that ALP activity of ALP fusion antibody obtained from cells cultured with a medium having a concentration of zinc chloride of 0.1mM is 100%.

(3) Results

As shown in fig. 6A, ALP activity was significantly low in the ALP fusion antibody obtained from the cells cultured with the medium without zinc addition. The results indicate that addition of zinc to the medium is necessary to exert ALP activity of the ALP fusion antibody. As shown in fig. 6B, even the ALP fusion antibody obtained from the cells cultured with the zinc supplemented medium after the expression of the ALP fusion antibody was significantly low in ALP activity. As a result, even if zinc was added to the medium at a period where it is considered that the ALP fusion antibody was expressed, the ALP activity of the ALP fusion antibody was not increased, indicating that zinc addition was necessary at the time of transfection.

As shown in FIG. 6C, ALP activity was sufficiently confirmed even when a medium containing 0.25mM zinc chloride was used. When a medium containing 0.5mM of zinc chloride was used, ALP activity could not be measured because cells were killed.

[ test example 3: consistency of ALP fusion antibody of example 1

The ALP fusion antibody of example 1 and the ALP-labeled antibody obtained by the chemical modification method were compared for the identity of the antibody molecules.

(1) Production of ALP-labeled antibody

Using plasmid DNA containing the gene of the rabbit anti-CD 80 antibody obtained in example 1, a rabbit anti-CD 80 antibody was obtained. Fab was obtained from the obtained antibody by a conventional method. The obtained Fab was combined with bovine small intestine-derived ALP (ALP55, ORIENTAL yeast) or recombinant ALP (rALP, Roche) using a cross-linking agent to obtain an ALP-labeled antibody.

(2) Size exclusion column chromatography (SEC) and SDS-PAGE

Each of the ALP fusion antibody (GS1 linker) and ALP labeled antibody of example 1 was separated by gel filtration using KTA avant25(GE Healthcare) and Superdex 200Increase 10/300GL (GE Healthcare). Gel filtration was performed in the same manner as in example 1. In addition, fractions of the ALP fusion antibody and the ALP-labeled antibody separated by gel filtration were analyzed by SDS-PAGE under non-reducing conditions.

(3) Results

The elution profiles of the gel filtration are shown in FIGS. 7A and B. The results of SDS-PAGE are shown in FIGS. 8A and B. In the figure, "Fab +" refers to a labeled antibody in which ALP of molecule 1 is bound to Fab of molecule 1, "Fab + +" refers to a labeled antibody in which ALP of molecule 1 is bound to Fab of molecule 2, and "Fab + +" refers to a labeled antibody in which ALP of molecule 1 is bound to Fab of molecule 3. Referring to FIG. 7A, in gel filtration, Fab + + + was eluted into 10 mL-10.5 mL, Fab + + was eluted into 11 mL-11.5 mL, and Fab + was eluted into 12 mL-12.5 mL. Referring to FIG. 7B, the ALP fusion antibody was eluted into a fraction of 12mL to 12.5mL in the gel filtration. As shown in fig. 7A, it was found that labeled antibodies with various molecular weights, unmodified ALPs, and Fab' were mixed in ALP labeled antibodies obtained by the chemical modification method. FIG. 8A also shows heterogeneity of ALP-labeled antibodies obtained by chemical modification. On the other hand, as shown in fig. 7B and 8B, a sharp single peak was observed in the elution profile of the ALP fusion antibody. Thus, the ALP fusion antibody of the present embodiment is a group of uniform molecules.

[ test example 4: properties of reagent containing ALP fusion antibody of example 1

A reagent used in a fully automatic immunoassay device was prepared using the ALP fusion antibody of example 1, and the performance of the reagent was examined. For comparison, reagents for each fraction using the ALP-labeled antibody isolated in experimental example 3 were also investigated.

(1) Preparation of reagent

The rabbit anti-CD 80 antibody (half IgG) was labeled with biotin by a conventional method and dissolved in a buffer for R1 reagent (50mM HEPES, 150mM NaCl, 1% BSA, pH 7.4) to prepare a R1 reagent (capture antibody reagent). As the R2 reagent (solid phase), HISCL R2 reagent (Sysmex) containing streptavidin-bound magnetic particles was used. Buffer for R3 reagent (50mM HEPES, 1mM MgCl)₂、0.1mM ZnCl₂1% BSA, pH 7.3) dissolved the ALP fusion antibody (GS1 linker) of example 1 to prepare a reagent R3 (detection antibody reagent). The concentration of ALP fusion antibody in the R3 reagent was 200 ng/mL. For comparison, R3 reagents containing various fractions (Fab +, Fab + + or Fab + + +) of ALP-labeled antibodies were similarly prepared. The concentration of the ALP-labeled antibody in the R3 reagent was adjusted in such a manner that the ALP activity of the R3 reagent containing the ALP fusion antibody became the same. As the R4 reagent (buffer for measurement), HISCL R4 reagent (Sysmex corporation) was used. As the R5 reagent (substrate solution), HISCL R5 reagent (Sysmex Co., Ltd.) was used. An antigen solution was prepared by diluting human CD80/B7-1 protein (His-tag) with BufferI' stage. As a cleaning solution for the magnetic particles, HISCL cleaning solution (Sysmex) was used.

(2) Measurement of

Using the above-mentioned reagent, measurement was carried out by a fully automatic immunoassay device HISCL-800 (manufactured by Sysmex Co., Ltd.). This assay is based on a sandwich ELISA on magnetic particles. Specific operations are as follows. After an antigen solution (20. mu.L) was added to and mixed with the R1 reagent (50. mu.L), the R2 reagent (30. mu.L) was added and mixed. The magnetic particles in the obtained mixture were collected to remove the supernatant, and HISCL cleaning solution (300. mu.L) was added to clean the magnetic particles. The supernatant was removed, and an R3 reagent (100. mu.L) was added to the magnetic particles, followed by mixing. The magnetic particles in the obtained mixture were collected to remove the supernatant, and HISCL cleaning solution (300. mu.L) was added to clean the magnetic particles. The supernatant was removed, and an R4 reagent (50. mu.L) and an R5 reagent (100. mu.L) were added to the magnetic particles, and the mixture was thoroughly mixed to measure the chemiluminescence intensity. In order to examine the background of the measurement, the measurement was performed in the same manner as described above except that BufferI' containing no antigen was used instead of the antigen solution. The measured SN ratio was calculated from the measured value and background of each antigen solution.

(3) Results

The measurement results are shown in fig. 9A and B. As shown in fig. 9A, the background was that the side of using the ALP fusion antibody as the detection antibody was significantly lower than that of using the ALP labeled antibody. As shown in fig. 9B, the SN ratio was significantly higher when the ALP fusion antibody was used as the detection antibody than when the ALP-labeled antibody was used. From these results, it was found that the antigen can be detected with high sensitivity by using the detection antibody reagent containing the ALP fusion antibody, as compared with the detection antibody reagent containing the ALP-labeled antibody obtained by the chemical modification method.

Example 2: preparation of bovine intestinal derived ALP fusion antibody (2)

As an antibody part, a fusion protein in which Fab and ALP subunits of a mouse anti-human IgG antibody were bound was prepared, using an ALP fusion antibody different from the ALP fusion antibody of example 1. This corresponds to the ALP fusion antibody having the structure shown in antibody example 1-1 described above.

(1) Preparation of expression vector

Lymphocytes were obtained from peripheral blood of mice immunized with human IgG, and mRNA was extracted from the lymphocytes to synthesize cDNA. Using the obtained cDNA, plasmid DNA containing the gene of a mouse anti-human IgG antibody was obtained in the same manner as in example 1. Using plasmid DNA containing the antibody gene and the BIAPII expression vector of example 1, an Fd-linker-BIAPII expression vector was obtained in the same manner as in example 1. The expression vector obtained had a gene encoding peptide linker GS1, GS3, EK1 or EK3 and BIAPII gene linked downstream of the gene encoding Fd of mouse anti-human IgG antibody. Further, a light chain expression vector was obtained in the same manner as in example 1 using plasmid DNA containing the gene of the mouse anti-human IgG antibody.

(2) Production and culture of antibody-producing cells

The above-mentioned expression vector was transfected into Expi293F (trade mark) cells cultured in a medium supplemented with a zinc chloride solution in the same manner as in example 1. The culture supernatant was recovered 5 days after the self-transfection, and the ALP fusion antibody was concentrated and purified in the same manner as in example 1.

[ test example 5: quality of reagent containing ALP fusion antibody of example 2

The molecular identities of the ALP fusion antibody obtained in example 2 and the ALP-labeled antibody obtained by the chemical modification method were compared. In addition, a reagent for use in a fully automatic immunoassay device was prepared using the ALP fusion antibody obtained in example 2, and the performance and storage stability of the reagent were examined. For comparison, a reagent for labeling an antibody using ALP was also discussed.

(1) Preparation of ALP-labeled antibody and confirmation of identity of antibody molecule

An ALP-labeled antibody was obtained by binding Fab of a mouse anti-human IgG antibody to ALP55(ORIENTAL Yeast Co., Ltd.) or rALP (Roche). Each of the ALP fusion antibody and ALP labeled antibody obtained in example 2 was separated by gel filtration in the same manner as in experimental example 3. In addition, fractions of the ALP fusion antibody and the ALP-labeled antibody separated by gel filtration were analyzed by SDS-PAGE under non-reducing conditions. As the results of gel filtration and SDS-PAGE, labeled antibodies having various molecular weights, unmodified ALP and Fab' were mixed in the ALP-labeled antibody obtained by the chemical modification method, similarly to the results of test example 3. In one aspect, the ALP fusion antibody is a homogeneous population of molecules.

(2) Preparation and measurement of reagent

In the same manner as in test example 4, an R1 reagent containing a biotin-labeled mouse anti-human IgG antibody (half IgG) and an R3 reagent containing the ALP fusion antibody obtained in example 2 were prepared. For comparison, an R3 reagent containing an ALP-labeled antibody of the fraction obtained by gel filtration was similarly prepared. As the fraction of ALP-labeled antibody, a fraction of labeled antibody bound to ALP containing 1 molecule and Fab containing 2 molecules (rALP-2Fab Fr.B4, rALP-2Fab Fr.B5, and ALP55-2Fab Fr.B4) and a fraction of labeled antibody bound to ALP containing 1 molecule and Fab containing 1 molecule (ALP55-1Fab Fr.B6) were used. The antibody concentration in each R3 reagent was adjusted based on the ALP activity of each antibody. An antigen solution was prepared by diluting a human IgG antibody with BufferI'. The R2 reagent, R4 reagent, R5 reagent and cleaning solution were the same as in test example 4. Using these reagents, measurement was carried out using HISCL-800 (manufactured by Sysmex Co., Ltd.), and the background and SN ratio of the measurement were calculated. The measurement results are shown in FIG. 10. In fig. 10, points denoted by ∑ represent the values of the background, and bars of the graph represent the SN ratios.

(3) Examination of storage stability of reagent

The measurement values were compared by measuring BufferI' without an antigen solution and an antigen with HISCL-800 (manufactured by Sysmex corporation) using the R3 reagent stored at 4 ℃ and the R3 reagent stored at 40 ℃ for 1 week. The results are shown in Table 1. In the table, "NC" indicates BufferI' containing no antigen, PC indicates an antigen solution, "count" indicates a measurement value, and "vs. 4 ℃" indicates a ratio of a measurement value obtained by the R3 reagent used for storage at 40 ℃ for 1 week to a measurement value obtained by the R3 reagent used for storage at 4 ℃.

[ TABLE 1 ]

(4) Results

As shown in fig. 10, the background is that the side when the ALP fusion antibody was used was significantly lower than when the ALP labeling antibody was used. The SN ratio was significantly higher when the ALP fusion antibody was used than when the ALP-labeled antibody was used. As shown in table 1, it was found that the measured value of PC in the R3 reagent containing the ALP fusion antibody was not much decreased from the measured value of PC in the R3 reagent containing the ALP labeling antibody when each R3 reagent was stored at 40 ℃ for 1 week. Thus, it was demonstrated that the reagent containing the ALP fusion antibody had higher storage stability than the reagent containing the ALP-labeled antibody.

Example 3: preparation of bovine intestinal derived ALP fusion antibody (3)

Using the plasmid DNA containing the antibody gene obtained in example 2 and the BIAPII expression vector, expression vectors having the structures shown in the above-mentioned configuration examples 1-2, 1-3, 2-1 and 2-2 were prepared in the same manner as in example 1. Using these expression vectors, ALP fusion antibodies shown in antibody examples 1-2, 1-3, 2-1 and 2-2 were prepared.

(1) Preparation of expression vector and obtaining of antibody

(1.1) ALP fusion antibody of antibody example 1-2

Plasmid DNA containing the antibody gene obtained in example 2 was used as a template to obtain a DNA fragment encoding a light chain and a peptide linker. The obtained DNA fragment was ligated with linearized BIAPII expression vector DNA to prepare an LC-linker-BIAPII expression vector. In this expression vector, a gene encoding a peptide linker and a BIAPII gene were ligated downstream of the gene encoding the light chain. Further, a DNA fragment encoding Fd was obtained by using plasmid DNA containing the above-described antibody gene as a template, and an expression vector containing the gene encoding Fd (Fd expression vector) was prepared in the same manner as in example 1. These expression vectors were transfected into Expi293F (trade mark) cells to obtain ALP fusion antibodies of antibody examples 1-2.

(1.2) ALP fusion antibody of antibody examples 1-3

Plasmid DNA containing the antibody gene obtained in example 2 was used as a template to obtain a DNA fragment encoding Fd. The obtained DNA fragment was ligated with linearized BIAPII expression vector DNA to prepare Fd-BIAPII expression vector. In this expression vector, a BIAPII gene is ligated downstream of the gene encoding Fd. The expression vector and the light chain expression vector obtained in example 2 were transfected into Expi293F (trademark) cells to obtain ALP fusion antibodies of antibody examples 1-3.

(1.3) ALP fusion antibody of antibody example 2-1

Plasmid DNA containing the antibody gene obtained in example 2 was used as a template to obtain a DNA fragment encoding the peptide linker and Fd. The obtained DNA fragment was ligated with linearized BIAPII expression vector DNA to prepare a BIAPII-linker-Fd expression vector. In this expression vector, a gene encoding a peptide linker and a gene encoding Fd are ligated downstream of the biapiii gene. This expression vector and the light chain expression vector obtained in example 2 were transfected into Expi293F (trade mark) cells to obtain the ALP fusion antibody of antibody example 2-1.

(1.4) ALP fusion antibody of antibody example 2-2

Plasmid DNA containing the antibody gene obtained in example 2 was used as a template to obtain a DNA fragment encoding a peptide linker and a light chain. The obtained DNA fragment was ligated with linearized BIAPII expression vector DNA to prepare a BIAPII-linker-LC expression vector. In this expression vector, a gene encoding a peptide linker and a gene encoding a light chain are ligated to the downstream of the BIAPII gene. The expression vector and the Fd expression vector described above were transfected into Expi293F (trade mark) cells to obtain an ALP fusion antibody of antibody example 2-2.

(2) Investigation of antibody molecule identity and reagent performance

Using each of the prepared ALP fusion antibodies, the identity of antibody molecules was examined in the same manner as in experimental example 3. The results of gel filtration are shown in FIGS. 11A-D. The ALP activity of each ALP fusion antibody was measured in the same manner as in experimental example 2. For comparison, an assay using the ALP fusion antibody of example 1 (antibody example 1-1) was also performed. The results are shown in FIG. 11E. An ELISA method was performed in the same manner as in test example 1 except that each of the ALP fusion antibodies of antibody examples 1-2, 1-3 and 2-1 obtained in the above (1) was used as a detection antibody in place of the ALP fusion antibody of example 1. For comparison, an assay using the ALP fusion antibody of example 1 (antibody example 1-1) was also performed. The results are shown in FIG. 11F.

Referring to FIGS. 11A to D, the ALP fusion antibodies of antibody examples 1-2, 1-3, 2-1 and 2-2 were eluted in fractions of 11mL to 11.5mL in the gel filtration. As shown in FIGS. 11A to D, the obtained ALP fusion antibody was a uniform molecular aggregate. In addition, as shown in fig. 11E, the obtained ALP fusion antibody had ALP activity. As shown in FIG. 11F, similarly to the ALP fusion antibody of antibody example 1-1, the ALP fusion antibodies of antibody examples 1-2, 1-3 and 2-1 each had an increased emission intensity depending on the antigen concentration. Thus, the ALP fusion antibodies of antibody examples 1-2, 1-3 and 2-1 all had antigen detectability.

Example 4: preparation of bovine intestinal derived ALP fusion antibody (4)

A fusion protein in which VHH and ALP subunits of an alpaca anti-CTLA-4 antibody were bound was prepared, and an ALP fusion antibody was obtained. This corresponds to the ALP fusion antibody having the structure shown in the above-mentioned antibody example 3-1.

(1) Preparation of expression vector and obtaining of antibody

A DNA fragment encoding VHH and a peptide linker (GS1 linker) was obtained by using plasmid DNA containing the gene encoding VHH of the alpaca anti-CTLA-4 antibody as a template. The obtained DNA fragment was ligated with linearized BIAPII expression vector DNA to prepare a VHH-linker-BIAPII expression vector. In this expression vector, a gene encoding a peptide linker and a BIAPII gene were ligated downstream of the gene encoding VHH. The ALP fusion antibody of antibody example 3-1 was obtained by transfecting VHH-linker-BIAPII expression vector into Expi293F (trade mark) cells.

(2) Measurement of ALP Activity and examination of antibody molecule identity

The ALP activity of the ALP fusion antibody was measured in the same manner as in experimental example 2. For comparison, ALP activity of BIAPII obtained by transfecting the biapiii expression vector of example 1 into Expi293F (trademark) cells was also measured. The results are shown in FIG. 12A. In addition, the identity of the antibody molecules was investigated by gel filtration as in test example 3. The dissolution profile is shown in fig. 12B. In gel filtration, the ALP fusion anti-CTLA 4 antibody of antibody example 3-1 was eluted into 12 mL-12.5 mL fractions. As shown in fig. 12A, the resulting ALP fusion antibody had ALP activity. As shown in fig. 12B, the obtained ALP fusion antibody was a uniform aggregate of molecules.

Example 5: preparation of bovine intestinal derived ALP fusion antibody (5)

As an antibody part, a fusion protein in which Fab and ALP subunits of a rabbit anti-PD-1 antibody, a rabbit anti-PD-L1 antibody, or a mouse anti-VEGF antibody were bound was prepared, which was an ALP fusion antibody different from the ALP fusion antibody of example 1. This corresponds to the ALP fusion antibody having the structure shown in antibody example 1-1 described above.

(1) Preparation of expression vector

An Fd-linker-BIAPII expression vector containing a gene encoding the Fd of the rabbit anti-PD-1 antibody or rabbit anti-PD-L1 antibody, and a light chain expression vector containing a gene encoding the light chain of the antibody were obtained in the same manner as in example 1, except that PD-1 or PD-L1 was used as the immunogen. Due to the antibody gene for the anti-PD-1 antibody, a plurality of clones were obtained, and an ALP fusion antibody was prepared for each clone. In addition, in the same manner as in example 2 except that VEGF was used as an immunogen, an Fd-linker-BIAPII expression vector containing a gene encoding Fd of a mouse anti-VEGF antibody and a light chain expression vector containing a gene encoding a light chain of the antibody were obtained. The peptide linker is the GS1 linker.

(2) Investigation of the identity of antibody molecules and measurement of ALP Activity

Similarly to test example 3, the molecular identity of each ALP fusion antibody was examined by gel filtration. The dissolution curves are shown in FIGS. 13A to C. ALP activity was measured in the same manner as in test example 2 using an ALP fusion anti-PD-1 antibody or an ALP fusion anti-PD-L1 antibody. The results are shown in FIGS. 14A and B. Referring to FIGS. 13A to C, the ALP fusion anti-PD 1 antibody, the ALP fusion anti-PD-L1 antibody and the ALP fusion anti-VEGF antibody were eluted into fractions of 11.5mL to 12mL in the gel filtration. As shown in FIGS. 13A to C, the resulting ALP fusion antibody was a uniform molecular aggregate. In addition, as shown in fig. 14A and B, the obtained ALP fusion antibody had ALP activity.

Example 6: preparation of fusion antibody against ALP derived from Shewanella (Shewanella) bacterium

An ALP fusion antibody different from the ALP fusion antibody of example 1 as an ALP moiety was prepared as a fusion protein in which Fab of a rabbit antibody and ALP derived from a bacterium belonging to the genus Shewanel la (Shewanel la) were combined. This corresponds to the ALP fusion antibody having the structure shown in antibody example 1-1 described above.

(1) Preparation of expression vector and obtaining of antibody

As an expression vector for the rabbit antibody, plasmid DNA containing the gene of the rabbit anti-CD 80 antibody of example 1 was used. As a T3-3AP expression vector, a plasmid DNA containing the T3-3AP gene was obtained by gene synthesis with GenScript, based on the amino acid sequence of ALP (T3-3AP) derived from the T3-3 strain described in U.S. Pat. No. 9,133,446. The amino acid sequence of T3-3AP and the base sequence encoding it are shown in SEQ ID NOS: 3 and 4, respectively. An Fd-linker-T33 AP expression vector was obtained in the same manner as in example 1, except that a T3-3AP expression vector was used in place of the BIAPII expression vector. Peptide linkers are the GS1, EK1 and EK3 linkers. The expression vector and the light chain expression vector obtained in example 1 were transfected into Expi293F (trademark) cells to obtain an ALP fusion antibody.

(2) Investigation of the identity of antibody molecules and measurement of ALP Activity

The molecular identity of each ALP fusion antibody was examined by gel filtration in the same manner as in experimental example 3. The dissolution curves are shown in FIGS. 15A to C. In gel filtration, the ALP fusion anti-CD 80 antibody eluted into a fraction of 11.5mL to 12 mL. In addition, ALP activity was measured in the same manner as in experimental example 2 using each ALP fusion antibody. For comparison, ALP activity of the ALP fusion antibody of example 1 (Fab-GS1-BIAPII) was also determined. The results are shown in FIG. 16. As shown in FIGS. 15A to C, the obtained ALP fusion antibody was a uniform molecular aggregate. In addition, as shown in FIG. 16, the ALP fusion antibody having T3-3AP also exhibited high ALP activity, as with the ALP fusion antibody having BIAPII.

(3) Immunoassays using ALP fusion antibodies

(3.1) ELISA method

An ELISA method was performed in the same manner as in test example 1 except that the ALP fusion antibody obtained in (1) above was used as a detection antibody instead of the ALP fusion antibody of example 1. For comparison, an assay using the ALP fusion antibody of example 1 was also performed. The results are shown in FIG. 17. As shown in fig. 17, similarly to the ALP fusion antibody having BIAPII, the ALP fusion antibody having T3-3AP increased the emission intensity according to the antigen concentration. Thus, the ALP fusion antibodies with 3-3AP all had antigen detection ability.

(3.2) immunoassay Using an automatic measuring apparatus

The measurement was performed in the same manner as in experimental example 4, except that the R3 reagent containing the ALP fusion antibody obtained in (1) above was used instead of the ALP fusion antibody of example 1. For comparison, an assay using the R3 reagent containing the ALP fusion antibody of example 1 was also performed. The results are shown in FIGS. 18A and B. As shown in FIG. 18A, in the measurement using the R3 reagent containing ALP fusion antibody having T3-3AP, the emission intensity increased according to the antigen concentration. As shown in fig. 18B, the measurement using the R3 reagent showed a high SN ratio.

[ reference example: search for ALP derived from Shewanella (Shewanella) bacterium other than T3-3AP ]

In addition to T3-3AP, Shewanella (Shewanell la) bacteria-derived ALP useful for the ALP fusion antibody was searched as follows. Shewanella (Shewanella) -derived ALP having an identity of 69% or more with the amino acid sequence of T3-3AP (excluding the signal sequence) was searched by the identity search program BLAST (NCBI). As a result, 21 ALPs shown in Table 2 were selected as bacteria derived ALP of the genus Shewanell la other than T3-3 AP.

[ TABLE 2 ]

The amino acid sequence of the ALP was clustered using sequence analysis soft GENETYX (registered trademark) (GENETYX corporation). 6 ALPs (S-AP1, S-AP2, S-AP3, S-AP4, S-AP5, and S-AP6) shown in Table 3 were selected based on the clustering results.

[ TABLE 3 ]

Code	No.	Bacterial strains	The accession No.
				S-AP1	1	Shewanella putrescentiae (Shewanella putrefeaciens) CN-32	WP_011788159.1
S-AP2	2	Shewanella mansion (Shewanella xiamenensis)	WP_037428906.1
				S-AP3	4	Shewanella frigidimarina (Shewanella frigidimarina)	WP_011636029.1
S-AP4	9	Shewanella livingstonensis (Shewanella livingstonensis)	WP_124729648.1
				S-AP5	12	Shewanella frigidimarina (Shewanella frigidimarina)	WP_101032902.1
S-AP6	16	Shewanella vesiculosa (Shewanella vesiculosa)	WP_124017315.1

The signal sequence of these ALPs was predicted by the Signal peptide prediction program SingalP-5.0(DTU Health Tech). Based on the amino acid sequence of these ALP genes, GenScript was assigned to gene synthesis to obtain plasmid DNAs containing the respective ALP genes. In subcloning, the signal sequence of luciferase was inserted in place of the predicted signal sequence. Expression vectors for S-AP1, S-AP2, S-AP3, S-AP4, S-AP5 and S-AP6 were transfected into Expi293F (trade mark) cells, and ALP was recovered and purified by gel filtration. Using the purified ALP, the ALP activity was measured in the same manner as in Experimental example 2. For comparison, ALP activity of T3-3AP was also determined. The results are shown in FIG. 19. As shown in FIG. 19, it was revealed that S-AP1, S-AP2, S-AP3, S-AP4, S-AP5 and S-AP6, although having lower activity than T3-AP, are promising candidates for ALP used in ALP fusion antibodies.

[ description of symbols ]

11. 21: reagent kit

12. 22: 1 st Container

13. 24: packing case

14. 25: attached document

23: 2 nd container

Sequence listing

<110> Sysmex Corporation

<120> alkaline phosphatase fusion antibody and method for producing same, and reagent for immunoassay and immunoassay method

<130> 19-053JP

<160> 36

<170> PatentIn version 3.5

<210> 1

<211> 480

<212> PRT

<213> cattle (Bos taurus)

<400> 1

Leu Ile Pro Ala Glu Glu Glu Asn Pro Ala Phe Trp Asn Arg Gln Ala

1 5 10 15

Ala Gln Ala Leu Asp Val Ala Lys Lys Leu Gln Pro Ile Gln Thr Ala

20 25 30

Ala Lys Asn Val Ile Leu Phe Leu Gly Asp Gly Met Gly Val Pro Thr

35 40 45

Val Thr Ala Thr Arg Ile Leu Lys Gly Gln Met Asn Gly Lys Leu Gly

50 55 60

Pro Glu Thr Pro Leu Ala Met Asp Gln Phe Pro Tyr Val Ala Leu Ser

65 70 75 80

Lys Thr Tyr Asn Val Asp Arg Gln Val Pro Asp Ser Ala Gly Thr Ala

85 90 95

Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Tyr Arg Thr Ile Gly Val

100 105 110

Ser Ala Ala Ala Arg Tyr Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu

115 120 125

Val Thr Ser Val Ile Asn Arg Ala Lys Lys Ala Gly Lys Ala Val Gly

130 135 140

Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Ala Tyr

145 150 155 160

Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Leu Pro Ala

165 170 175

Asp Ala Gln Lys Asn Gly Cys Gln Asp Ile Ala Ala Gln Leu Val Tyr

180 185 190

Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Met Tyr Met Phe

195 200 205

Pro Glu Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Ala Ser Val Asn

210 215 220

Gly Val Arg Lys Asp Lys Gln Asn Leu Val Gln Glu Trp Gln Ala Lys

225 230 235 240

His Gln Gly Ala Gln Tyr Val Trp Asn Arg Thr Ala Leu Leu Gln Ala

245 250 255

Ala Asp Asp Ser Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Ala

260 265 270

Asp Met Lys Tyr Asn Val Gln Gln Asp His Thr Lys Asp Pro Thr Leu

275 280 285

Ala Glu Met Thr Glu Ala Ala Leu Gln Val Leu Ser Arg Asn Pro Arg

290 295 300

Gly Phe Tyr Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His His

305 310 315 320

Asp Gly Lys Ala Tyr Met Ala Leu Thr Glu Ala Ile Met Phe Asp Asn

325 330 335

Ala Ile Ala Lys Ala Asn Glu Leu Thr Ser Glu Leu Asp Thr Leu Ile

340 345 350

Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Thr

355 360 365

Leu Arg Gly Thr Ser Ile Phe Gly Leu Ala Pro Gly Lys Ala Leu Asp

370 375 380

Ser Lys Ser Tyr Thr Ser Ile Leu Tyr Gly Asn Gly Pro Gly Tyr Ala

385 390 395 400

Leu Gly Gly Gly Ser Arg Pro Asp Val Asn Gly Ser Thr Ser Glu Glu

405 410 415

Pro Ser Tyr Arg Gln Gln Ala Ala Val Pro Leu Ala Ser Glu Thr His

420 425 430

Gly Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His Leu

435 440 445

Val His Gly Val Gln Glu Glu Thr Phe Val Ala His Ile Met Ala Phe

450 455 460

Ala Gly Cys Val Glu Pro Tyr Thr Asp Cys Asn Leu Pro Ala Pro Ala

465 470 475 480

<210> 2

<211> 1440

<212> DNA

<213> cattle (Bos taurus)

<400> 2

ttaattccgg cagaagaaga aaacccggcg ttttggaacc gtcaggccgc acaggccctg 60

gatgtagcta aaaaacttca accgattcaa actgcagcca agaatgtgat cctgtttcta 120

ggggatggta tgggggtccc gacagtgaca gctactcgta ttttgaaggg tcagatgaat 180

gggaagttgg gcccggaaac ccctcttgca atggatcaat tcccttatgt ggctctttca 240

aagacatata atgttgatcg tcaagtacca gatagcgcag gtactgccac cgcgtactta 300

tgcggggtga aaggtaatta tcgtaccatt ggcgtatcgg cagctgctcg ctataaccag 360

tgtaatacca cccgtggaaa cgaagtgacc agcgtcatta accgcgcaaa gaaggcgggc 420

aaagcagtcg gcgtagtaac aacaactcgt gttcaacatg ctagcccggc tggcgcatac 480

gcacatacgg tcaatcgcaa ctggtattcg gatgccgacc ttccggctga tgctcaaaaa 540

aatggttgtc aagacatcgc ggcccaactt gtatacaata tggatatcga tgtcatcctt 600

ggcggcggcc gtatgtacat gtttcccgaa ggaaccccag atccggaata ccctgacgat 660

gcgtcggtga acggggtgcg taaagataaa cagaacttgg tccaagagtg gcaggccaaa 720

caccagggag ctcagtatgt ctggaatcga accgcgctgt tgcaggcagc agatgattcc 780

tcggttaccc acttaatggg acttttcgaa ccggccgata tgaaatacaa tgtccagcag 840

gaccacacaa aagaccctac cctggccgaa atgactgagg ccgcattaca agtcttaagc 900

cgtaacccca gagggttcta tctgttcgtt gagggcggcc gcatcgatca tgggcaccat 960

gatggcaaag cctatatggc ccttactgaa gccatcatgt tcgacaacgc gatagcgaag 1020

gcaaatgaat taacttctga actggatacc ctgatcttgg tgacagccga tcatagtcat 1080

gtattctcat ttgggggcta caccctgcgt ggtaccagta tctttggctt ggccccgggt 1140

aaggctctgg attccaagag ctatacctcc attctgtacg gtaacgggcc tgggtatgct 1200

cttgggggcg gttcgcgccc cgatgttaac ggttcaacgt ccgaagaacc ttcttatcgt 1260

cagcaggcag ccgtgcccct cgcgagcgag acgcatggag gtgaggacgt ggcggtgttt 1320

gctcgcggtc cgcaggcgca tcttgtacac ggagtgcaag aggagacgtt tgtagctcat 1380

ataatggcgt tcgcaggttg cgtggagcct tacacagatt gtaacttgcc tgcacctgcg 1440

<210> 3

<211> 443

<212> PRT

<213> Shewanella sp T3-3

<400> 3

Met Ser Val Thr Lys Thr Ser Leu Leu Leu Leu Thr Ile Gly Leu Val

1 5 10 15

Phe Ser Ala Ser Ser Lys Ala Ala Pro Glu Leu Glu Asn Gly Pro Met

20 25 30

Lys Pro Pro Ser Lys Pro Lys Asn Ile Val Ile Met Val Gly Asp Gly

35 40 45

Met Gly Pro Ser Tyr Thr Ser Ala Tyr Arg Tyr Phe Lys Asp Asn Pro

50 55 60

Asp Thr Glu Glu Val Glu Gln Thr Val Phe Asp Arg Leu Leu Val Gly

65 70 75 80

Met Ala Ser Thr Tyr Pro Ala Ser Val Ser Gly Tyr Val Thr Asp Ser

85 90 95

Ala Ala Ala Ala Thr Ala Leu Ala Thr Gly Val Lys Ser Tyr Asn Gly

100 105 110

Ala Ile Ser Val Asp Thr Gln Lys Gln His Leu Pro Thr Met Leu Glu

115 120 125

Lys Ala Lys Ala Leu Gly Leu Ser Thr Gly Val Ala Val Thr Ser Gln

130 135 140

Ile Asn His Ala Thr Pro Ala Ala Phe Leu Ala His Asn Glu Ser Arg

145 150 155 160

Lys Asn Tyr Asp Ala Leu Ala Leu Ser Tyr Leu Asp Thr Asn Ala Asp

165 170 175

Val Leu Leu Gly Gly Gly Gln Lys Tyr Phe Ser Pro Glu Leu Leu Glu

180 185 190

Lys Phe Thr Ala Lys Gly Tyr Gln His Ile Ser Arg Phe Glu Asp Leu

195 200 205

Ala Thr Ile Thr Gln Pro Lys Val Ile Gly Leu Phe Ala Gln Val Gln

210 215 220

Leu Pro Trp Ala Leu Asp Glu Lys Asn Ala Asn Arg Leu Ser Thr Met

225 230 235 240

Thr Gln Lys Ala Leu Asp Leu Leu Ser Gln Asn Glu Gln Gly Phe Val

245 250 255

Leu Leu Val Glu Gly Ser Leu Ile Asp Trp Ala Gly His Ser Asn Asp

260 265 270

Ile Ala Asn Thr Met Gly Glu Met Asp Glu Phe Ala Asn Ala Leu Glu

275 280 285

Val Val Glu Gln Phe Val Arg Gln His Pro Asp Thr Leu Met Val Ala

290 295 300

Thr Ala Asp His Asn Thr Gly Gly Leu Ser Ile Gly Ala Gly Gly Asp

305 310 315 320

Tyr Arg Trp Asn Pro Glu Ile Leu Arg Asn Met Ser Ala Ser Thr Asp

325 330 335

Thr Leu Ala Leu Ala Ala Leu Gly Gly Asp Gln Trp Gln Ala Asp Leu

340 345 350

Ala Arg Gly Leu Gly Phe Glu Leu Asn Ala Asp Glu Val Thr Gln Leu

355 360 365

Ser Thr Ala Arg Met Gln Gly Leu Glu Thr Met Thr Glu Ala Ile Arg

370 375 380

Lys Ile Ile Asp Lys Arg Thr Gly Thr Gly Trp Thr Thr Ser Gly His

385 390 395 400

Thr Gly Thr Asp Val Gln Val Phe Ala Ala Gly Pro Ala Ala Glu Leu

405 410 415

Phe Asn Gly His Gln Asp Asn Thr Asp Ile Ala Asn Lys Ile Phe Thr

420 425 430

Leu Leu Pro Lys Pro Lys Lys Ala Lys Thr Glu

435 440

<210> 4

<211> 1332

<212> DNA

<213> Shewanella sp T3-3

<400> 4

atgagcgtca ccaaaacatc actcttattg ctgactatcg gattagtatt ttcagctagc 60

agcaaggccg cacccgagct tgaaaacggg cctatgaaac cgccatcaaa acctaaaaac 120

atcgttatta tggtgggtga cggcatgggc ccttcgtaca ccagcgccta ccgctatttc 180

aaagataatc ctgacaccga agaagtcgaa caaaccgtat tcgatagact cttagttggc 240

atggcaagta cgtatcctgc cagtgtcagc ggctatgtca cagattctgc tgcggcggca 300

actgcgctcg ccacaggcgt aaaatcttat aatggcgcta tttccgtcga tacccaaaag 360

caacacttac caaccatgct cgaaaaagcc aaagcattag ggttaagcac aggtgtggcg 420

gtaacatcac aaatcaacca tgccacgccc gcggcatttt tagcccacaa cgagagccgt 480

aaaaattacg atgctctggc gctcagttat ttagacacaa atgccgatgt acttttgggc 540

ggcggacaga agtatttctc gcctgaactg ctcgaaaaat tcaccgccaa aggttatcaa 600

cacattagcc gctttgaaga tttggccact ataacccaac ccaaagtcat tggcctgttt 660

gcacaggtgc aactgccttg ggcgctcgat gagaaaaatg caaatcgcct cagcactatg 720

actcaaaaag ccctcgattt actctcacaa aatgagcaag gctttgtatt gttagtcgaa 780

ggcagcttga ttgactgggc cggacacagc aatgatatcg ccaacaccat gggcgaaatg 840

gatgaatttg ccaatgcact cgaagtggtt gagcagtttg tacgccaaca tccagacacc 900

ttaatggtag ccactgccga tcataatacc ggtggactct caattggtgc tggcggagat 960

tatcgctgga acccagagat tttacgcaat atgtctgcca gcacggacac gcttgcctta 1020

gccgcactcg gtggtgacca atggcaagcc gatctggccc gaggtttagg atttgagcta 1080

aacgccgatg aagtgactca attgagcaca gcccgaatgc aaggtcttga aaccatgact 1140

gaagccattc gtaaaatcat cgacaagcgc accggcactg gctggacaac ctcaggccac 1200

actggcacag acgtacaagt atttgccgca ggccctgctg ccgagttatt taatggccac 1260

caagataata ccgacatagc caacaaaatt ttcactttat tgcctaaacc gaaaaaagcc 1320

aaaaccgaat aa 1332

<210> 5

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 5

Gly Gly Gly Gly Ser

1 5

<210> 6

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 6

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 7

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 7

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 8

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 8

Glu Ala Ala Ala Lys

1 5

<210> 9

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 9

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10

<210> 10

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 10

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10 15

<210> 11

<211> 220

<212> PRT

<213> Rabbit (Oryctolagus cuniculus)

<400> 11

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asn Asn Ala

20 25 30

Val Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Ile Gly

35 40 45

Thr Ile Ser Ser Asn Asp Asn Thr Tyr Tyr Ala Thr Trp Ala Lys Gly

50 55 60

Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr

65 70 75 80

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Ile Tyr Tyr

85 90 95

Tyr Gly Tyr Gly Tyr Val Phe Asn Leu Trp Gly Pro Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu

130 135 140

Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly

145 150 155 160

Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln

180 185 190

Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp

195 200 205

Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr

210 215 220

<210> 12

<211> 213

<212> PRT

<213> Rabbit (Oryctolagus cuniculus)

<400> 12

Glu Leu Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly

1 5 10 15

Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Asp Ile Tyr Ser Ser

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Met

35 40 45

Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys

65 70 75 80

Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Tyr Leu Ser Ser Ser

85 90 95

Ser Arg Tyr Gly Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp

115 120 125

Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr

130 135 140

Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr

145 150 155 160

Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr

165 170 175

Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser

180 185 190

His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val

195 200 205

Gln Ser Phe Ile Gly

210

<210> 13

<211> 660

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 13

cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60

tgcacagcct ctggattctc cctcagtaat aatgcagtga gctgggtccg ccaggctcca 120

ggggaggggc tggaatggat cggaaccatt agtagtaatg ataacacata ttatgcgacc 180

tgggcgaaag gccgattcac catctccaaa acctcgacca cggtggatct gaaaatcacc 240

agtccgacaa ccgaggacac ggccacctat ttctgtgcca tctattatta tggttatggt 300

tatgttttta acttgtgggg cccaggcact ctggtcaccg tctcctcagg gcaacctaag 360

gctccatcag tcttcccact ggccccctgc tgcggggaca cacccagctc cacggtgacc 420

ctgggctgcc tggtcaaagg ctacctcccg gagccagtga ccgtgacctg gaactcgggc 480

accctcacca atggggtacg caccttcccg tccgtccggc agtcctcagg cctctactcg 540

ctgagcagcg tggtgagcgt gacctcaagc agccagcccg tcacctgcaa cgtggcccac 600

ccagccacca acaccaaagt ggacaagacc gttgcgccct cgacatgcag caagcccacg 660

<210> 14

<211> 650

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 14

gctatcgcag cgagctcgtg atgacccaga ctccagcctc cgtgtctgag cctgtgggag 60

gcacagtcac catcaattgc caggccagtg aggatattta cagctcttta gcctggtatc 120

agcagaaacc agggcagcgt cccaagctcc ttatgtatta tgcatccact ctggcatctg 180

gggtcccatc gcggttcaaa ggcagtggat ctgggacaga gttcactctc accatcagcg 240

acctggagtg tgccgatgct gccacttact actgtcaaag ctattatctt agtagtagta 300

gtaggtatgg taatgcgttc ggcggaggga ccgaggtggt ggtcaaaggt gatccagttg 360

cacctactgt cctcatcttc ccaccagctg ctgatcaggt ggcaactgga acagtcacca 420

tcgtgtgtgt ggcgaataaa tactttcccg atgtcaccgt cacctgggag gtggatggca 480

ccacccaaac aactggcatc gagaacagta aaacaccgca gaattctgca gattgtacct 540

acaacctcag cagcactctg acactgacca gcacacagta caacagccac aaagagtaca 600

cctgcaaggt gacccagggc acgacctcag tcgtccagag cttcataggg 650

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 15

tgataaaagg gttcgatccc tacc 24

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 16

gcagtgcacg gtggcgcagt acacc 25

<210> 17

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 17

gccaccgtgc actgcttaat tccggcagaa gaagaaaacc 40

<210> 18

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 18

cgaacccttt tatcacgcag gtgcaggcaa gttacaatc 39

<210> 19

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 19

ggtggcggtg gatccttaat tccggcagaa gaagaaaacc 40

<210> 20

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 20

gtggatccgg agggggcgga agtttaattc cggcagaaga agaaaacc 48

<210> 21

<211> 55

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 21

ggagggggcg gaagtggcgg gggaggttca ttaattccgg cagaagaaga aaacc 55

<210> 22

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 22

gaagccgctg ctaagttaat tccggcagaa gaagaaaacc 40

<210> 23

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 23

ctgctaagga ggcagccgcg aaattaattc cggcagaaga agaaaacc 48

<210> 24

<211> 55

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 24

gaggcagccg cgaaagaagc agcggctaaa ttaattccgg cagaagaaga aaacc 55

<210> 25

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 25

gcagtgcacg gtggcgcagt acacc 25

<210> 26

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 26

gccaccgtgc actgccagtc ggtggaggag tccgg 35

<210> 27

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 27

ggatccaccg ccacccgtgg gcttgctgca tgtcgaggg 39

<210> 28

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 28

ccccctccgg atccaccgcc acccgtgggc ttgctgcatg tcgaggg 47

<210> 29

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 29

acttccgccc cctccggatc caccgccacc cgtgggcttg ctgcatgtcg aggg 54

<210> 30

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 30

cttagcagcg gcttccgtgg gcttgctgca tgtcgaggg 39

<210> 31

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 31

gctgcctcct tagcagcggc ttccgtgggc ttgctgcatg tcgaggg 47

<210> 32

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 32

tttcgcggct gcctccttag cagcggcttc cgtgggcttg ctgcatgtcg aggg 54

<210> 33

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 33

taatctagat aattaaaggg ttcg 24

<210> 34

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 34

gctgcgatag cccggaaaca gtacc 25

<210> 35

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 35

ccgggctatc gcagcgagct cgtgatgacc cagac 35

<210> 36

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 36

taattatcta gattatcaac agtcacccct attgaagc 38