JP2003319789A - Protein capable of binding to female sex hormone and process for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide proteins capable of binding to female sex hormones which have useful properties added thereto, for example, having high sensitivity in assaying, quantifying or concentrating the female sex hormones, showing little cross-reactivity, being scarcely affected by interfering substances and being scarcely affected by solvents. <P>SOLUTION: The recombinant proteins are produced by obtaining various antibody genes against the female sex hormones and then modifying various characteristics, for example, affinity for an antigen, binding properties to an antigen, cross reactivity, tolerance to a substance interfering an antigen-antibody reaction, tolerance to a substance interfering an enzymatic color development reaction, tolerance to a solvent, etc., of the original antibodies by genetically modification techniques. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody for female hormones, a gene encoding the same, a protein capable of binding to the female hormones and a method for producing the same, a method for measuring or quantifying female hormones, and a method for determining female hormones. Concentration method, etc.

[0002]

2. Description of the Related Art In recent years, environmental pollution due to female hormones has become a problem. It is necessary to measure and analyze female hormones and their degradation products in the environment and use the results for environmental conservation. Several excellent methods are known as such measurement and analysis methods (for example, Patent Document 1).
reference).

[0003]

[Patent Document 1] Japanese Patent Laid-Open No. 2001-41958

[0004]

DISCLOSURE OF THE INVENTION The present invention obtains various antibody genes against female hormones, and has affinity for the antigen of the original antibody, antigen-binding ability, cross-reactivity, resistance to antigen-antibody reaction interfering substances, Cross-reaction with good sensitivity in measuring, quantifying and concentrating female hormones to recombinant proteins obtained by creating various properties such as enzyme color reaction interfering substance resistance and solvent resistance by modified technology of gene manipulation The present invention intends to prepare and utilize a protein having a binding ability to female hormones, which has useful properties such as low activity, less influence of interfering substances, and less influence of solvent. Examples of female hormones include estrogens such as estradiol (E2), estrone (E1), and estriol (E3), ethinyl estradiol (EE2), and diethylbestrol (DE).
And synthetic female hormones such as S).

[0005]

[Means for Solving the Problems] The inventors of the present invention have earnestly studied to obtain a protein having the ability to bind to female hormones, which has useful properties such as sensitivity and measurable by improving affinity. Then, create a transformant carrying the gene or modified gene,
As a result of further research and finding that a protein having the ability to bind to female hormones can be efficiently produced, the present invention was completed. That is, the present invention provides (1) the following protein [a] or [b] or a salt thereof: [a] the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 6, and SEQ ID NO: 10 The amino acid sequence represented by, the amino acid sequence represented by SEQ ID NO: 14, the amino acid sequence represented by SEQ ID NO: 18,
Alternatively, a protein having substantially the same amino acid sequence as these (including the heavy chain variable region of an anti-female hormone antibody); [b] the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 8 , An amino acid sequence represented by SEQ ID NO: 12, an amino acid sequence represented by SEQ ID NO: 16, an amino acid sequence represented by SEQ ID NO: 20, or an amino acid sequence substantially identical to these (anti-female hormones) Protein containing the variable region of the antibody light chain). (2) The following [a ^1] - [a ^3], either protein or a salt thereof [b ^1] - [b ^3]: [a ^1] or less (i) the amino acid sequence of any of - (v) And the specific region is selected from the group consisting of complementarity determining region 1, complementarity determining region 2, complementarity determining region 3, framework region 1, framework region 2, framework region 3, and framework region 4. A protein characterized by: (i) an amino acid sequence corresponding to one or more specific regions in the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 6; Exchange with an amino acid sequence represented by SEQ ID NO: 10, an amino acid sequence represented by SEQ ID NO: 14, or an amino acid sequence corresponding to one or more specific regions of the same type contained in the amino acid sequence represented by SEQ ID NO: 18. Has been Amino acid sequence, (ii) of the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence corresponding to one or more specific regions is represented by the amino acid sequence represented by SEQ ID NO: 2, represented by SEQ ID NO: 10 An amino acid sequence, an amino acid sequence represented by SEQ ID NO: 14 or an amino acid sequence exchanged with an amino acid sequence corresponding to one or more specific regions of the same type contained in the amino acid sequence represented by SEQ ID NO: 18, (iii) ) Among the amino acid sequence represented by SEQ ID NO: 10, the amino acid sequence corresponding to one or more specific regions is the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 6, the SEQ ID NO: Is replaced with an amino acid sequence corresponding to one or more specific regions of the same kind contained in the amino acid sequence represented by: 14 or the amino acid sequence represented by SEQ ID NO: 18. (Iv) the amino acid sequence represented by SEQ ID NO: 14, the amino acid sequence corresponding to one or more specific regions is represented by SEQ ID NO: 2, Amino acid sequence, SEQ ID NO:
An amino acid sequence represented by 10 or an amino acid sequence exchanged with an amino acid sequence corresponding to one or more specific regions of the same type contained in the amino acid sequence represented by SEQ ID NO: 18, or (v) SEQ ID NO: 18 In the amino acid sequence represented by, the amino acid sequence corresponding to one or more specific regions is the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO:
The amino acid sequence represented by 6 or the amino acid sequence represented by SEQ ID NO: 10 or the amino acid sequence represented by SEQ ID NO: 14 has been replaced with an amino acid sequence corresponding to one or more specific regions of the same type. Amino acid sequence: [b ¹ ] consisting of the amino acid sequence of any of (i) to (v) below, wherein the specific regions are complementarity determining region 1, complementarity determining region 2, complementarity determining region 3, framework region 1 A protein selected from the group consisting of :, framework region 2, framework region 3, and framework region 4, (i) one or more of the amino acid sequences represented by SEQ ID NO: 4 The amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 16. Or (ii) of the amino acid sequence represented by SEQ ID NO: 8, which is exchanged with an amino acid sequence corresponding to one or more specific regions of the same type contained in the amino acid sequence represented by SEQ ID NO: 20. Among them, the amino acid sequence corresponding to one or more specific regions is the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 16, or SEQ ID NO: An amino acid sequence exchanged with an amino acid sequence corresponding to one or more specific regions of the same type contained in the amino acid sequence represented by 20, (iii) one or more of the amino acid sequence represented by SEQ ID NO: 12 The amino acid sequence corresponding to the specific region is the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 8, or the amino acid sequence represented by SEQ ID NO: 16. Or an amino acid sequence exchanged with an amino acid sequence corresponding to one or more specific regions of the same type contained in the amino acid sequence represented by SEQ ID NO: 20, (iv) among the amino acid sequence represented by SEQ ID NO: 16 The amino acid sequence corresponding to one or more specific regions is the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO:
Amino acid sequence represented by 12 or the amino acid sequence represented by SEQ ID NO: 20, which is replaced with an amino acid sequence corresponding to one or more specific regions of the same type, or (v) SEQ ID NO: 20 In the amino acid sequence represented by, the amino acid sequence corresponding to one or more specific regions is the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO:
The amino acid sequence represented by 8 or the amino acid sequence represented by SEQ ID NO: 12 or the amino acid sequence represented by SEQ ID NO: 16 is replaced with an amino acid sequence corresponding to one or more specific regions of the same type. Amino acid sequence; [a ² ] amino acid sequence represented by SEQ ID NO: 2, amino acid sequence represented by SEQ ID NO: 6, amino acid sequence represented by SEQ ID NO: 10, amino acid sequence represented by SEQ ID NO: 14 , An amino acid sequence represented by SEQ ID NO: 18 or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the protein of [a ¹ ] and SEQ ID NO: 4 The amino acid sequence represented by, the amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 16, A protein that binds to female hormones when forming a complex with a protein having the amino acid sequence represented by SEQ ID NO: 20 or the amino acid sequence of the protein of [b ¹ ]; [b ² ] SEQ ID NO: 4 The amino acid sequence represented by, the amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 16, the amino acid sequence represented by SEQ ID NO: 20. Alternatively, it has an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the protein of [b ¹ ] and has the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 6 The amino acid sequence represented by, the amino acid sequence represented by SEQ ID NO: 10, the amino acid sequence represented by SEQ ID NO: 14, the amino acid sequence represented by SEQ ID NO: 18, or A protein that binds to female hormones when forming a complex with a protein having the amino acid sequence of the protein of [a ¹ ]; [a ³ ] the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 6 The amino acid sequence represented by SEQ ID NO: 10, the amino acid sequence represented by SEQ ID NO: 14, the amino acid sequence represented by SEQ ID NO: 18, or the amino acid sequence of the protein of [a ¹ ] An amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 8, or an amino acid sequence represented by SEQ ID NO: 12, which has an amino acid sequence in which one or more amino acids are deleted, substituted or added The amino acid sequence represented by SEQ ID NO: 16, the amino acid sequence represented by SEQ ID NO: 20, or the amino acid sequence of the protein of [b ¹ ] Or a protein which binds to female hormones when forming a complex with a protein having an amino acid sequence in which two or more amino acids are deleted, substituted or added; [b ³ ] represented by SEQ ID NO: 4 Amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 12, amino acid sequence represented by SEQ ID NO: 16, amino acid sequence represented by SEQ ID NO: 20, or b ¹ ] having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of the protein [ ¹ ], and the amino acid sequence represented by SEQ ID NO: 2 is represented by SEQ ID NO: 6. Amino acid sequence represented by SEQ ID NO: 10, amino acid sequence represented by SEQ ID NO: 14, amino acid sequence represented by SEQ ID NO: 18, or the amino acid sequence of [a ¹ ] A protein that binds to female hormones when forming a complex with a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the protein. (3) The protein or salt thereof according to (1) or (2) above, wherein the female hormone is estrogen or a synthetic female hormone. (4) The protein or salt thereof according to (3) above, wherein the estrogen is estradiol, estrone or estriol. (5) The protein or a salt thereof according to (3) above, wherein the synthetic female hormone is ethinyl estradiol or diethyl bestrol. (6) A method for gene recombination of the protein according to any one of (1) to (5) above. (7) A protein or a salt thereof obtained by the method described in (6) above. (8) A partial peptide of the protein according to any one of (1) to (5) and (7) above or a salt thereof. (9) A polynucleotide encoding the protein or the partial peptide thereof according to any one of (1) to (5) and (7) above. (10) A recombinant vector containing the polynucleotide according to (9) above. (11) A transformant transformed with the recombinant vector according to (10) above. (12) The protein according to any one of (1) to (5) and (7) above, a partial peptide thereof, or a salt thereof is produced and collected, which is characterized in (1) above. ~ A method for producing the protein according to any one of (5) and (7), a partial peptide thereof, or a salt thereof. (13) A complex formed by linking the protein of [a] below with the protein of [b] below: [a] Amino acid sequence represented by SEQ ID NO: 2, amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: : The amino acid sequence represented by: 10, the amino acid sequence represented by SEQ ID NO: 14, the amino acid sequence represented by SEQ ID NO: 18,
Or a protein having an amino acid sequence substantially the same as these; [b] the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence represented by SEQ ID NO: 12, the SEQ ID NO: A protein having the amino acid sequence represented by 16, the amino acid sequence represented by SEQ ID NO: 20, or an amino acid sequence substantially the same as these. (14) The complex according to (13) above, which is a single-chain antibody. (15) A phage which expresses the single chain antibody according to (14) above. (16) Use the complex described in (13) or (14) above (or the protein described in any one of (1) to (5) and (7) above), or the phage described in (15) above. A method for identifying female hormones that bind to the complex, comprising: (17) The complex according to (13) or (14) (or the protein according to any one of (1) to (5) and (7)), or the phage according to (15) is used. A method for measuring or quantifying female hormones, which comprises: (18) The complex according to (13) or (14) (or the protein according to any one of (1) to (5) and (7)) or the phage according to (15). , A kit for measuring or quantifying female hormones. (19) The complex according to (13) or (14) (or the protein according to any one of (1) to (5) and (7)), or the phage according to (15) is used. A method for concentrating female hormones, which comprises: (20) The complex according to (13) or (14) (or the protein according to any one of (1) to (5) and (7)), or the phage according to (15). , A kit for concentrating female hormones. (21) E2-73 strain (FERM BP-7569),
E1-420 strain (FERM BP-7568), EE2
-227 strain (FERM BP-7567), EE2-3
A mouse hybridoma cell which is a strain or an EE2-8-18 strain. (22) A monoclonal antibody produced by the mouse hybridoma cell according to the above (21).

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION [a] The amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 10,
Amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 1
A protein having (or consisting of) the amino acid sequence represented by 8 or an amino acid sequence substantially the same as these,
Or [b] the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 12
A protein having (or consisting of) the amino acid sequence represented by, the amino acid sequence represented by SEQ ID NO: 16, the amino acid sequence represented by SEQ ID NO: 20, or an amino acid sequence substantially identical to these is provided. . The protein of [a] above is a protein having the amino acid sequence of the heavy chain variable region of an anti-female hormone antibody or an amino acid sequence substantially the same as that, while the protein of [b] above is an anti-female hormone. It is a protein having an amino acid sequence of a light chain variable region of an antibody or an amino acid sequence substantially the same as that. Hereinafter, description will be made in order.

The protein [a] is not particularly limited as long as it has the amino acid sequence of the heavy chain variable region of the anti-female hormone antibody or an amino acid sequence substantially the same as the heavy chain variable region, but in one embodiment, The protein of [a] is
¹ ], [a ² ] and [a ³ ] proteins. Also,
The protein [a ¹ ] has the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 6,
Amino acid sequence represented by SEQ ID NO: 10, SEQ ID NO: 1
The amino acid sequence represented by 4 or the amino acid sequence represented by SEQ ID NO: 18 is not particularly limited as long as it is a protein consisting of an amino acid sequence in which a specific region is replaced with a specific region of another amino acid sequence. Is from (i) above
A protein consisting of the amino acid sequence of (v) can be mentioned.

As the specific area in the above [a ¹ ],
Complementarity determining region 1, complementarity determining region 2, complementarity determining region 3 (hereinafter referred to as CDR1, CDR2, CDR3 as necessary.
And framework region 1, framework region 2, framework region 3, and framework region 4 (hereinafter, abbreviated as FR1, FR2, FR3, FR4 as necessary). In [a ¹ ] above, the amino acid sequences to be exchanged are preferably amino acid sequences of the same kind of specific region. Therefore, for example, an amino acid sequence corresponding to CDR1 in the amino acid sequence represented by SEQ ID NO: 6 and a CD in the amino acid sequence represented by SEQ ID NO: 2
A protein having an amino acid sequence corresponding to R2, CDR3, FR1, FR2, FR3, FR4 is provided. The number of specific areas to be exchanged is not particularly limited as long as it is 1 or more, but for example, 1 to 3, preferably 1 to 2,
More preferably, it is one. The exchange of amino acid sequences can be performed by a method known per se. Specifically, a primer is designed such that the portions corresponding to the exchanged regions are connected to the primers corresponding to the N and C ends of each region, and the fragment is amplified by PCR using this primer, PCR may be performed with a combination that has been replaced anew.

SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO:
10, CDR1, CDR2, CDR3, F in the amino acid sequence represented by SEQ ID NO: 14 or SEQ ID NO: 18
Specifically, the regions corresponding to R1, FR2, FR3, and FR4 are as follows: (i) CDR1 (25th to 34th amino acid residues in the amino acid sequence represented by SEQ ID NO: 2) , 25th to 34th amino acid residues in the amino acid sequence represented by SEQ ID NO: 6, 25th to 36th amino acid residues in the amino acid sequence represented by SEQ ID NO: 10, and SEQ ID NO: 14 25th to 34th amino acid residues in the amino acid sequence shown, and 25 in the amino acid sequence shown in SEQ ID NO: 18.
(Amino acid residue from the thirty-sixth position); (ii) CDR2 (49th to the 58th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2;
Amino acid residues 49 to 59 in the amino acid sequence represented by SEQ ID NO: 6, amino acid residues 51 to 59 in the amino acid sequence represented by SEQ ID NO: 10, represented by SEQ ID NO: 14 Amino acid residues 49 to 57 in the amino acid sequence,
And 5 in the amino acid sequence represented by SEQ ID NO: 18.
(1) to 59th amino acid residues); (iii) CDR3 (from the 96th to 106th amino acid residues in the amino acid sequence represented by SEQ ID NO: 2, in the amino acid sequence represented by SEQ ID NO: 6) 98
The amino acid residue from the 10th position to the 106th position, SEQ ID NO: 1
99 to 107 in the amino acid sequence represented by 0
Amino acid residues up to the th, amino acid residues 97 to 107 in the amino acid sequence represented by SEQ ID NO: 14, and amino acid residues 99 to 107 in the amino acid sequence represented by SEQ ID NO: 18. Group); (iv) FR1 (amino acid residues 1 to 24 in the amino acid sequence represented by SEQ ID NO: 2, 1 to 2 in the amino acid sequence represented by SEQ ID NO: 6)
Amino acid residues up to the 4th, amino acid residues 1 to 24 in the amino acid sequence represented by SEQ ID NO: 10, amino acid residues 1 to 24 in the amino acid sequence represented by SEQ ID NO: 14 Group and 1 to 2 in the amino acid sequence represented by SEQ ID NO: 18
4th amino acid residue); (v) FR2 (from the 35th to 48th amino acid residues in the amino acid sequence represented by SEQ ID NO: 2, from the 35th amino acid sequence represented by SEQ ID NO: 6) Amino acid residues up to 48th, amino acid residues 37 to 50 in the amino acid sequence represented by SEQ ID NO: 10, amino acid residues 35 to 48 in the amino acid sequence represented by SEQ ID NO: 14 Group and amino acid residues 37 to 50 in the amino acid sequence represented by SEQ ID NO: 18); (vi) FR3 (59 to 95 amino acids in the amino acid sequence represented by SEQ ID NO: 2) Residues, 60th to 97th amino acid residues in the amino acid sequence represented by SEQ ID NO: 6, represented by SEQ ID NO: 10 That amino acid residues from the 60th in the amino acid sequence up to 98th, SEQ ID NO: amino acid residues from position 58 in the amino acid sequence represented by 14 to 96 th, and SEQ ID NO: 18 60 in the amino acid sequence represented by
(Amino acid residues from the 98th to 98th); (vii) FR4 (amino acid residues from 107 to 117 in the amino acid sequence represented by SEQ ID NO: 2, 10 in the amino acid sequence represented by SEQ ID NO: 6)
Amino acid residues 7 to 118, SEQ ID NO:
108th to 1st in the amino acid sequence represented by 10
The 18th amino acid residue, the 108th to 118th amino acid residues in the amino acid sequence represented by SEQ ID NO: 14, and the 108th to 118th amino acids in the amino acid sequence represented by SEQ ID NO: 18. residue).

[0010] In another embodiment, the protein of [a] above is, for example, [a ⁴ ] SEQ ID NO: 2, SEQ ID NO:
6, SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 1
8 having an amino acid sequence represented by 8 or an amino acid sequence having significant homology to the amino acid sequence of the protein of [a ¹ ] above, and having SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, Female hormones when formed into a complex with a protein having an amino acid sequence represented by SEQ ID NO: 16 or SEQ ID NO: 20 or an amino acid sequence having significant homology to the amino acid sequence of the protein [b] above Can be a protein that binds to.

The protein [b] is not particularly limited as long as it has the amino acid sequence of the light chain variable region of the anti-female hormone antibody or an amino acid sequence substantially the same as that, but in one embodiment, The protein of [b] is
¹ ], [b ² ] and [b ³ ] proteins. Also,
The protein of [b ¹ ] above has the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 8,
The amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 1
The amino acid sequence represented by 6 or the amino acid sequence represented by SEQ ID NO: 20 is not particularly limited as long as it is a protein consisting of an amino acid sequence in which a specific region is replaced with a specific region of another amino acid sequence. Is from (i) above
A protein consisting of the amino acid sequence of (v) can be mentioned.

As the specific area in the above [b ¹ ],
CDR1, CDR2, CDR3, FR1, FR2, FR
3 and FR4. In [b ¹ ] above, the amino acid sequence to be exchanged is preferably the amino acid sequence of the same kind of specific region. Therefore, for example, an amino acid sequence corresponding to CDR1 in the amino acid sequence represented by SEQ ID NO: 4 and CDR2, CDR3, FR1, FR2, FR3, FR in the amino acid sequence represented by SEQ ID NO: 8
A protein having an amino acid sequence corresponding to 4 is provided. The number of specific areas to be replaced is not particularly limited as long as it is 1 or more, but for example, 1 to 3, preferably 1 to
Two, more preferably one.

SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO:
12, CDR1, CDR2, CDR3, F in the amino acid sequence represented by SEQ ID NO: 16 or SEQ ID NO: 20
The regions corresponding to R1, FR2, FR3, and FR4 are specifically as follows: (i) CDR1 (24th to 34th amino acid residues in the amino acid sequence represented by SEQ ID NO: 4) In the amino acid sequence represented by SEQ ID NO: 8, amino acid residues 24 to 39, in the amino acid sequence represented by SEQ ID NO: 12, amino acid residues 24 to 40, in SEQ ID NO: 16 24th to 34th amino acid residues in the amino acid sequence represented, and 24 in the amino acid sequence represented by SEQ ID NO: 20
Amino acid residues from the 40th to 40th); (ii) CDR2 (50th to 56th amino acid residues in the amino acid sequence represented by SEQ ID NO: 4;
Amino acid residues 55 to 60 in the amino acid sequence represented by SEQ ID NO: 8, amino acid residues 56 to 62 in the amino acid sequence represented by SEQ ID NO: 12, represented by SEQ ID NO: 16 50th to 56th amino acid residues in the amino acid sequence,
And 5 in the amino acid sequence represented by SEQ ID NO: 20.
(6th to 62nd amino acid residues); (iii) CDR3 (89th to 97th amino acid residues in the amino acid sequence represented by SEQ ID NO: 4, in the amino acid sequence represented by SEQ ID NO: 8) 94
Amino acid residues from position 102 to SEQ ID NO: 1
95 to 102 in the amino acid sequence represented by 2
Amino acid residues up to the th position, amino acid residues 89 to 97 in the amino acid sequence represented by SEQ ID NO: 16, and amino acid residues 95 to 102 in the amino acid sequence represented by SEQ ID NO: 20 Group); (iv) FR1 (amino acid residues 1 to 23 in the amino acid sequence represented by SEQ ID NO: 4, 1 to 2 in the amino acid sequence represented by SEQ ID NO: 8)
Amino acid residues up to the third, amino acid residues 1 to 23 in the amino acid sequence represented by SEQ ID NO: 12, amino acid residues 1 to 23 in the amino acid sequence represented by SEQ ID NO: 16 Group and 1 to 2 in the amino acid sequence represented by SEQ ID NO: 20
(V) FR2 (amino acid residues from the 35th position to the 49th position in the amino acid sequence represented by SEQ ID NO: 4, from the 40th position in the amino acid sequence represented by SEQ ID NO: 8) Amino acid residues up to the 54th, amino acid residues 41 to 55 in the amino acid sequence represented by SEQ ID NO: 12, amino acid residues 35 to 49 in the amino acid sequence represented by SEQ ID NO: 16 Group, and amino acid residues 41 to 55 in the amino acid sequence represented by SEQ ID NO: 20); (vi) FR3 (amino acids 57 to 88 in the amino acid sequence represented by SEQ ID NO: 4) Residues, amino acid residues 61 to 93 in the amino acid sequence represented by SEQ ID NO: 8, represented by SEQ ID NO: 12 That amino acid residues from 63 th in the amino acid sequence up to 94th, SEQ ID NO: amino acid residues 16 to 57 th in the amino acid sequence represented by up to 88 th, and SEQ ID NO: 63 in the amino acid sequence represented by 20
(Amino acid residues from the 94th to 94th); (vii) FR4 (98th to 108th amino acid residues in the amino acid sequence represented by SEQ ID NO: 4, 10 in the amino acid sequence represented by SEQ ID NO: 8)
Amino acid residues 3 to 113, SEQ ID NO:
103 to 1 in the amino acid sequence represented by 12
The 13th amino acid residue, the 98th to 107th amino acid residues in the amino acid sequence represented by SEQ ID NO: 16, and the 103rd to 113th amino acids in the amino acid sequence represented by SEQ ID NO: 20 residue).

In another embodiment, the protein of [b] above is, for example, [b ⁴ ] amino acid sequence represented by SEQ ID NO: 4, amino acid sequence represented by SEQ ID NO: 8, or SEQ ID NO: Amino acid sequence represented by 12, amino acid sequence represented by SEQ ID NO: 16, amino acid sequence represented by SEQ ID NO: 20, or amino acid having significant homology to the amino acid sequence of the protein of [b ¹ ] above The amino acid sequence having a sequence and represented by SEQ ID NO: 2, SEQ ID NO:
6, the amino acid sequence represented by SEQ ID NO: 10, the amino acid sequence represented by SEQ ID NO: 14, the amino acid sequence represented by SEQ ID NO: 18, or the amino acid sequence of the protein of [a] above. It may be a protein that binds to female hormones when it forms a complex with a protein having an amino acid sequence having significant homology to.

In the present invention, the number of amino acids to be deleted, substituted or added in the amino acid sequence represented by any SEQ ID NO: X is not particularly limited as long as it is 1 or 2 or more. It may be 80, preferably about 1 to 20, more preferably about 1 to 9, even more preferably 1 to 5, and most preferably several (1 or 2).

In the present invention, the amino acid substitution is not particularly limited as long as the particular amino acid is replaced with any other amino acid, and may be, for example, a conservative amino acid substitution or a non-conservative amino acid substitution. The "conservative amino acid substitution" refers to substitution of a specific amino acid with an amino acid having a side chain having a property similar to that of the amino acid. Specifically, in a conservative amino acid substitution, a particular amino acid is replaced with another amino acid that belongs to the same group as that amino acid. On the other hand, “non-conservative amino acid substitution” means substitution of a specific amino acid with an amino acid having a side chain having a property different from that of the amino acid. Specifically, in a non-conservative amino acid substitution, a specific amino acid is replaced with another amino acid belonging to a group different from that amino acid. Groups of amino acids with side chains of similar nature are known in the art. For example, such groups of amino acids include amino acids having basic (ie, positively charged) side chains (eg, lysine, arginine, histidine), acidic (ie, negatively charged) side chains. Amino acids with chains (eg aspartic acid, glutamic acid), amino acids with neutral (ie uncharged) side chains (eg glycine, asparagine, glutamine, serine, threonine,
Tyrosine, cysteine, alanine, valine, leucine,
Isoleucine, proline, phenylalanine, methionine, tryptophan). Further, the amino acid having a neutral side chain, further, an amino acid having a polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), and an amino acid having a non-polar side chain (for example, alanine, Valine, leucine, isoleucine, proline, phenylalanine,
Methionine, tryptophan). Further, as another group, for example, an amino acid having an aromatic side chain (for example, phenylalanine, tryptophan, histidine), a hydroxyl group (alcoholic hydroxyl group,
An amino acid having a side chain containing a phenolic hydroxyl group (for example, serine, threonine, tyrosine) and the like can also be mentioned.

The amino acid sequence having significant homology to the amino acid sequence represented by any SEQ ID NO: X is, for example, about 40 amino acid sequences represented by any SEQ ID NO: X. % Or more, preferably 60% or more,
More preferred is an amino acid sequence having a homology of about 80% or more, even more preferably about 90% or more, and most preferably about 95% or more.

The degree of homology (%) can be determined by a method known per se. For example, the degree of homology (%) is determined by the algorithm of Smith and Waterman (Adv. App
L. Math., 1981, 2, 482-489) (Wisconsin Sequence Analysis Package, Version)
8 for Unix (registered trademark), Genetics Computer Group, U
niversity Research Park, Madison WI) by default. Also, Karl
in and Altschul's algorithm (Proc. Natl. Acad. Sc
i. USA, 1990, 87: 2264-2268, Proc. Natl. Acad. Sci.
USA, 1993, 90: 5873-5877) may be used. For example, when comparing the homology of proteins, the degree of homology (%) can be determined by using the XBLAST program by default. In addition, Myers and Miller (CABIOS, 1988, 4: 11-1
The ALIGN program (vers
ion 2.0) (GCG sequence alignment software package
May be used). Settings for comparing amino acid sequences using the ALIGN program include, for example, PAM120.
weight residue table, gap length penalty = 12, gap
penalty = 4. Also, these programs can be used in the same manner to determine the degree of homology (%) of base sequences.

"Binds to female hormones when forming a complex" means that the complex is reactive to female hormones. The female hormones are not particularly limited as long as they bind to an estrogen receptor (eg, estrogen receptor α, estrogen receptor β) and exhibit an estrogen-like action, and examples thereof include estrogen and synthetic female hormones. Of these, estradiol, estrone, estriol, ethinyl estradiol and diethyl bestrol are preferred. Whether or not the complex has the ability to bind to female hormones can be determined by a method known per se such as the measuring method described below or a method similar thereto. Incidentally, the complex of the present invention,
It should have the ability to bind to any of the above female hormones.

In one embodiment, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO:
Amino acid sequence represented by 8, amino acid sequence represented by SEQ ID NO: 10, amino acid sequence represented by SEQ ID NO: 12, amino acid sequence represented by SEQ ID NO: 14, amino acid represented by SEQ ID NO: 16 Sequence, a protein having the amino acid sequence represented by SEQ ID NO: 18 or the amino acid sequence represented by SEQ ID NO: 20, or the above [a ¹ ] and [b ¹ ]
[A ² ] to [a ⁴ ] in which the binding ability and cross-reactivity to female hormones have been changed by introducing a deletion, substitution or addition of one or more amino acids into the protein of
The proteins [b ² ] to [b ⁴ ] can be obtained. Regions in which one or more amino acids are deleted, substituted or added in the proteins [a ² ] to [a ⁴ ] and [b ² ] to [b ⁴ ] are CDR1, CDR2, CDR3, FR1, FR2,
It may be one or more regions selected from the group consisting of FR3 and FR4.

The partial peptide of the present invention is not particularly limited as long as it is a peptide constituting a part of the protein of [a] or [b] above. For example, the partial peptide of the above [a] or [b] above In the amino acid sequence, a peptide composed of at least 6 or more, preferably at least 8 or more, more preferably at least 10 or more, even more preferably at least 12 or more, most preferably 15 or more consecutive amino acids is used. Further, as the partial peptide of the present invention, CDR1, CDR2, CDR3, F of the above-mentioned protein [a] or the above-mentioned protein [b] is used.
A partial peptide having (or consisting of) an amino acid sequence corresponding to R1, FR2, FR3, FR4 can also be used.

As the salt of the protein of the present invention or its partial peptide, a salt known per se, such as an acid addition salt, can be used. Examples of acid addition salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

In the present invention, the "complex" is not particularly limited as long as the protein [a] above and the protein [b] above are linked. For example, the protein [a] above and the above [b] ] The protein of the above] is covalently bound to the protein via a linker or not. Further, the complex can be used in the form of a salt (preferably an acid addition salt) like the above-mentioned protein and partial peptide.

The linker used for fusing the protein of the above [a] and the protein of the above [b] may be any known in the art and is not particularly limited. For example, GGGS (SEQ ID NO: : 27) repeat sequence, GSTSGSGKSSEGKG (SEQ ID NO: 56), GSTSGSGKSSEG
SGSTKG (SEQ ID NO: 57), GSTSGKPSEGKG (SEQ ID NO:
58), GSTSGSGKPGSGEGSTST (SEQ ID NO: 59) and the like can be used as a linker (for example, Production of single-chain Fv monomers and mul).
timers, D. Filpula, J. McGuire, and M. Whitlow. In
"Antibody Engineering" Edited by J. McCafferty,
HR Hoogenboon, and DJ Chiswell.pp.253-268,
See IRL PRESS (1996)). The complex in which the above-mentioned protein [a] and the above-mentioned protein [b] are covalently bonded with or without a linker is, for example, the above-mentioned [a] protein and the above-mentioned [b] protein separately. After the preparation, these proteins can be obtained by covalently bonding each to a linker, or by directly covalently bonding without a linker. However, this method is complicated because it requires a step of linking the protein of the above [a] and the protein of the above [b] in order to obtain a complex. In addition, there is a possibility that a plurality of substances having different covalent bond sites may be obtained, and it is difficult to prepare a preferable single complex from the viewpoint of reproducibility and the like. Therefore, the complex of the present invention includes, for example, a single-chain antibody in which the above-mentioned protein [a] and the above-mentioned protein [b] are fused by amide bond via a peptide linker or by direct amide bond. (In the present specification, it may be referred to as a recombinant antibody, a single chain variable region antibody, a single chain antibody or the like). The single-chain antibody includes the base sequence encoding the protein of [a] above, the base sequence encoding the peptide linker (when obtaining an amide-bonded single chain antibody through the linker), the protein of [b] above. It is useful because it can be easily prepared from a transformant containing an expression vector containing the nucleotide sequence coding for and the reading frame. The base sequence encoding the peptide linker is not particularly limited as long as it does not contain a stop codon when the reading frames are aligned with the base sequences encoding the proteins [a] and [b].

The peptide linker can be appropriately selected by a method known in the art. Specifically, as the peptide linker, a peptide consisting of one or more amino acid residues and having an arbitrary length can be used. For example, a peptide consisting of 10 or more amino acid residues is used.

The present invention also provides a polynucleotide encoding the amino acid sequence of the protein of the present invention (in the present specification, synonymous with gene and DNA). The polynucleotide of the present invention may be any one as long as it contains the nucleotide sequence encoding the protein of the present invention described above.

Specifically, as the polynucleotide of the present invention, the nucleotide sequence encoding the protein of the above [a] (eg, SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13 or A base sequence represented by SEQ ID NO: 17) and a base sequence encoding the protein of the above [b] (for example, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15 or SEQ ID NO: 19) Represented nucleotide sequence),
Examples include the nucleotide sequence encoding the above single-chain antibody.
The polynucleotide of the present invention may also include a polynucleotide having a nucleotide sequence encoding a protein obtained by genetically recombining the protein of the present invention.

The present invention also provides a phage characterized by expressing the single chain antibody of the present invention. The phage of the present invention is not particularly limited as long as it expresses the single chain antibody of the present invention, but preferably the single chain antibody of the present invention is displayed on the surface of the phage. As the phagemid, a phagemid capable of displaying the single chain antibody of the present invention on the phage surface can be used. For example, pCANTAB 5E
(Amersham Pharmacia Biotech), p8V5 (Affyma
x company, etc. (for example, Vectors for Phage D)
isplay, N. Armstrong, NB Adey, SJ McConnell,
and BK Kay, in "Phage Display of Peptides and P
roteins "Edited by BK Kay, J. Winter, and J. Mc
See Cafferty. Pp.35-53, Academic Press (1996)). As the helper phage, a helper phage that can be used in a host transformed with the above phagemid can be used, and examples thereof include M13KO7 (Amersham Pharmacia Biotech) and other M13-based helper phages.

The above-mentioned gene (DNA, polynucleotide) of the present invention can be obtained by a known method based on the disclosure of the present specification. For example, but not limited to, the gene of the present invention can be obtained from a hybridoma producing an anti-female hormone monoclonal antibody. First, determine the N-terminal amino acid sequence of the antibody protein,
Then, a primer having a base sequence deduced from this amino acid sequence is prepared, mRNA is prepared from the antibody-producing hybridoma by a known method, and single-stranded cDNA is synthesized based on the mRNA by reverse transcriptase. Based on the amino acid sequence or base sequence of the variable region of the heavy chain or light chain of the disclosed anti-female hormone monoclonal antibody, P
The gene of the present invention can be selectively obtained by using the CR method, the hybridization method, or the like. Such methods are well known, and those skilled in the art can easily isolate the gene of the present invention based on the disclosure of the present specification. Specific operating methods of these methods include, for example, molecular cloning (M
olecular Cloning) 3rd edition (J. Sambrook et.al., C
old Spring Harbor Lab. Press, 2001) and the like. In addition, mRNA extraction was performed by Amersham Qu
The method described in the operation manual of the ickPrep mRNA purification kit can be mentioned, and cDNA synthesis and 5'-RACE method are performed by Slon of Clontech.
Examples include the method described in the MART RACE kit operation manual.

Recombinant Antibodi
es), please refer to RECOMBINANT ANTIBOD
IES (ed.by F. Breitling, John Wiley & Sons (USA), 199
9), Chapter 2, Recombinant Antibody Fragments (Recombinant Antibody
Fragments), hybridoma cells (Hybrid
Cloning of the antibody gene from the oma cell line (Clo
ning) method, antibody gene library (Antibody Gene
Libraries), selection of recombinant antibodies from gene libraries (Selection of Recombinant Antibodies
From Gene Libraries) method, gene manipulation of antibodies (Anti
body engineering) methods are described, and recombinant antibodies can be produced by these methods.

In Chapter 4 of the same book, a method for producing a recombinant antibody or a recombinant antibody fragment is also described. In vitro expression in rabbit Reticulocyte lysate is
ryote), Cytoplasm of E. coli, periplas
solubule fraction of m, inclusion body of periplasm
And expression in Bacillus and Streptomyces,
karyote), Pichia, Saccharomyces, Schizosaccha
Yeasts such as romyces, molds such as Trichoderma, Baculovirus in mycelia cells, animal cells such as myeloma, CHO, COS, transgenic plants such as tobacco, teransgenic animals, etc. have been described the expression method, by these transformants It can be manufactured.

Furthermore, Chapter 4 of the same book describes a method for purifying a recombinant antibody or a fragment of the recombinant antibody. The target product is separated by cell disruption by sonic waves, mechanical abrasion, or enzymatic lysis. Next, ion exchange chromatography, size exclusion chromatography, thiophili
c adsorption chromatography, affinity chromatograp
Purify by combining hy, etc. Especially affinity chromar
Tography is an efficient method, and it uses antigen-apecific methods that utilize antigen recognition specificity, protein A and protein G.
Antibodies utilizing binding to Fc and Fab 'sites such as
specific method or in the case of scFv that does not have such a site, it is expressed as a fusion antibody with a small peptide fragment called tag, and a method using an affinity column specific to this tag (eg Hia-tag, c -myc tag, Strep tag
Etc.) and the like.

First, a cDNA library of anti-female hormone monoclonal antibody-producing cells was constructed, and a cDNA encoding the N-terminal sequences of the constant and variable regions of the heavy and light chains of immunoglobulin with high conservation was probed. The cDNA library can be screened to isolate the light chain and heavy chain cDNAs of the anti-female hormone monoclonal antibody.

Specific operation methods of these methods include:
For example, Molecular Cloni
ng) 3rd edition (J. Sambrook et.al., Cold Spring Har
borLab. Press, 2001).
The gene of the present invention may also be chemically synthesized using well-known techniques based on the sequences described herein.

The method of gene recombination of the protein of the present invention may be a method known per se, for example, a method of converting the nucleotide sequence encoding the protein can be used. Conversion of the nucleotide sequence of DNA is carried out using PCR or a known kit such as Mutan ^™ -Super Express Km (Takara Shuzo Co., Ltd.) or Mutan ^™ -K (Takara Shuzo Co., Ltd.).
-LAPCR method, Gupped duplex method, Kunkel method or the like known per se or a method analogous thereto can be used. The cloned DNA encoding the antibody protein can be used as it is, or if desired after digestion with a restriction enzyme or addition of a linker, depending on the purpose. The DNA may have ATG as a translation initiation codon at the 5'-terminal side and TAA, TGA or TAG as a translation termination codon at the 3'-terminal side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor. The expression vector of the antibody protein of the present invention is, for example,
(A) It can be produced by cutting out a target DNA fragment from the DNA encoding the antibody protein of the present invention and (b) ligating the DNA fragment downstream of a promoter in an appropriate expression vector.

Methods for Producing Recombinant Antibodies Although various forms of recombinant antibodies can be produced, Roland Kontermann's ANTIBODY ENGINEERING HOME P
AGE (http://aximt1.imt.uni-marburg.de/~rek/AEP.htm
l, February 25, 2002) are examples thereof, for example, Fab 'fragments, F (ab') fragments, Fv fragmen.
ts (Fv), single-chain Fv fragments (scFv), bispeci
fic-chimeric scFV (χ-scFv), tandem scFV (scFv) 2,
bispecific- (scFv) 2, disulfide-linked scFv, disulfi
de-stabilized Fv fragments (dsFv), diabody, single-
chain diabody (scDb), bivalent diabody, bispecific
diabody, knob-into-hole stabilized diabody, disul
fide-stabilized diabody, triabody, tetrabody, tris
pecific triabody, CL-dimerized scFv, CH1-CL-dimeri
zed scFv, CH3-dimerized scFv, knob-into-hole CH3-d
imerized scFv, CH3-dimerized bivalent diabody, Fc-
dimerized scFv, Fab-scFv fusions, Ig-scFv fusions,
leucine-zipper stabilized scFv dimers, helix-stabi
lized scFv dimers, 4 helix-bunde stabilized scFv t
It can be prepared as a recombinant antibody such as etramers, streptavidin-scFv, and intrabody. Further, a method for selecting an antibody having a desired useful property by shuffling the antibody gene subjected to mutation treatment is also within the scope of the present invention.

Recombinant Antibody Expression System Any recombinant antibody expression system may be used as long as it can efficiently express the recombinant antibody.
nd Kontermann's ANTI BODY ENGINEERING HOME PAGE (htt
p: //aximt1.imt.uni-marburg.de/~rek/AEP.html, 2002
(February 25, 2013), for example, mammalia
In n cells, Fv, scFv, scFv derivatives, bivalent
And bispecific scFv, scFv or Fab-fusion protei
Expression of ns, intrabodies, etc.
Expression of V, Fab, etc. is found in Fv, scFv and Fa in Fungal cells.
Expression of scFv is known to be expressed in Plants cells such as expression of b, and thus various expression systems can be used.

Method for preparing cDNA library As a method for preparing a cDNA library, cD can be efficiently prepared.
Any method can be used as long as it can create an NA library, but Roland Kontermann's ANTI BODY ENGINE
ERING HOME PAGE (http://aximt1.imt.uni-marburg.de/
The phage display method described in ~ rek / AEP.html, February 25, 2002) is one of them.

Method for selecting recombinant antibody As a method for selecting a desired recombinant antibody from the prepared library, Roland Kontermann's ANTI BODY ENGINE
ERING HOME PAGE (http://aximt1.imt.uni-marburg.de/
~ rek / AEP.html, February 25, 2002), such as the "isolation method of recombinant antibody from phagemid library" and "isolation method of peptide from fd phage library" Methods can also be used as selection methods.

The gene (DNA) used for expression has substantially the same amino acid sequence as the anti-female hormone antibody protein expressed by the gene (DNA) obtained above, or is a partial peptide thereof. But it's okay. The substantially identical amino acid sequence means a part of the amino acid sequence that has been modified (substituted), an amino acid sequence that has been added, an amino acid sequence that has been deleted (deleted), etc. Can be mentioned. The gene (DNA) can be used as it is, or can be used by cutting or adding other DNA as desired depending on the purpose. The DN
A may have a translation initiation codon ATG at the end. It can be carried out by a method known per se. For example, Molecular Cloning
3rd edition (J. Sambrook et. Al., Cold Spring Harbo
r Lab. Press, 2001).

A recombinant vector can be produced by incorporating the DNA (polynucleotide) thus obtained into a vector with a promoter, a translation initiation codon, an appropriate signal sequence and the like by a method known per se. it can. Examples of the vector, promoter, and host strain include molecular cloning (Molecular Cloni
ng) 3rd Edition (J. Sambrook et.al., Cold Spring Harbor L
ab. Press, 2001), vectors, promoters, Escherichia strains and the like.

As the vector, in addition to the above, a plasmid (pET-276, pCANTAP-5) derived from Escherichia coli is used.
E, pUC19, pT7BuleT) Bacillus subtilis-derived plasmid (eg, pUB110, pTP5, pC194),
Yeast-derived plasmid (eg, pSH19, pSH1
5), λ phage, bacteriophage such as M13K07, animal viruses such as retrovirus, vaccinia virus, baculovirus, pA1-11,
pXT1, pRc / CMV, pRc / RSV, pcDN
AI / Neo or the like is used.

As a promoter, used for gene expression
If the promoter is appropriate for the host
Anything is fine. For example, if the host is Escherichia
In the case of, the trp promoter, the lac promoter,
recA promoter, λP _LPromoter, lpp
If the host is Bacillus, such as Lomotor,
SPO1 promoter, SPO2 promoter, pen
When the host is yeast such as P promoter, PHO
5 promoter, PGK promoter, GAP promotion
And the ADH promoter are preferred. Host is animal
In the case of cells, SRα promoter, SV40 pro
Motor, LTR promoter, CMV promoter,
The host is an insect cell such as HSV-TK promoter
In case of polyhedrin promoter, P10 promoter
ー are preferred.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin and the like can be used in addition to the above. As a selection marker, for example, an ampicillin resistance gene (hereinafter Am
p ^R ), kanamycin resistance gene (hereinafter sometimes referred to as Km ^R ), chloramphenicol resistance gene (hereinafter sometimes referred to as Cm ^R )
Etc. If necessary, a signal sequence suitable for the host is added to the N-terminal side of the antibody protein of the present invention. If the host is a genus Escherichia, phoA
Signal sequence, ompA signal sequence, etc., when the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc., when the host is yeast, MFα signal sequence, SUC2 signal When the host is an animal cell such as a sequence, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, etc. can be used, respectively. Using the vector containing the DNA encoding the antibody protein of the present invention thus constructed, transformants can be produced.

As the host, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia spp. Include Escherichia coli K12 DH1 [Procedures of the National Academy of
Sciences of the USA (Proc. Nat
L. Acad. Sci. USA), Volume 60, 160 (1968)],
JM103 [Nucleic Acids Research, (nuc
leic Acids Research), Volume 9, 309 (1981)],
JA221 [Journal of Molecular Biology], Volume 120,
517 (1978)], HB101 [Journal of Morecue Biology, 41, 459 (196).
9), C600 [Genetics, 39
Vol., 440 (1954)], BL21DE3 (pLys
S), TG-1, JM109, etc. are used. Examples of the bacterium of the genus Bacillus include Bacillus subtilis (Baci
llus subtilis) MI114 [Gene, 24 volumes,
255 (1983)], 207-21 [Journal of Biochemistry,
95, 87 (1984)] and the like. Examples of yeast include Saccharomyces cerevisiae (Sacch
^{aromyces cerevisiae) AH22, AH22R -,} NA8
7-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NC
YC1913, NCYC2036, Pichia pastoris
(Pichia pastoris) and the like are used. Examples of insect cells include Spodoptera frugiperda cell (Sf) when the virus is AcNPV.
Cells), MG1 cells derived from the midgut of Trichoplusia ni, Tri
High Five ^TM cells derived from eggs of choplusia ni, Mamestrabra
Cells derived from ssicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyxmori N; BmN cell) or the like is used. Examples of the Sf cells include Sf9 cells (ATCC
CRL1711), Sf21 cells (above Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like are used. As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 5].
92 (1985)]. As animal cells, for example, monkey cells COS-7, Vero, Chinese hamster cells CHO, mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like can be used.

To transform Escherichia, for example, Proceedings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69 Vol, 2110 (1
972) and Gene, Volume 17, 107 (198).
It can be performed according to the method described in 2) or the like. To transform Bacillus, for example, molecular
And General Genetics (Molecular &
General Genetics), 168, 111 (1979) and the like. For transforming yeast, for example, Methods in Enzymology, 194, 182-
187 (1991), Procedures of the National Academy of Sciences of
The USA (Proc.Natl.Acad.Sci.USA), 75
Vol., 1929 (1978) and the like. For transforming insect cells or insects, for example, Bio / Technolog
y), Vol. 6, 47-55 (1988) and the like. For transforming animal cells, for example, the method described in Cell Engineering Supplement 8, New Cell Engineering Experimental Protocol, 263-267 (1995) (published by Shujunsha), Virology, Volume 52, 456 (1973). Can be done according to. Thus, a transformant transformed with the expression vector containing the DNA encoding the antibody protein can be obtained. further,
By culturing the transformant thus obtained, the protein of the present invention is produced, and by collecting this, the protein of the present invention can be produced.

As the medium used for culture, a liquid medium is suitable as the medium used when the transformant whose host is the genus Escherichia or the bacterium Bacillus is cultivated. It contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. As the carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc., as the nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, etc. Inorganic or organic substances, as the inorganic substance, for example,
Examples thereof include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast, vitamins, growth promoting factors and the like may be added. The pH of the medium is about 5
8 is desirable.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York (19
72)] is preferred. If necessary, a drug such as 3β-indolylacrylic acid or isopropylthiogalactoside (IPTG) can be added to the promoter so that the promoter works efficiently. When the host is a bacterium of the genus Escherichia, the culture is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is Bacillus, the culture is usually about 30-4.
It is carried out at 0 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant whose host is yeast, examples of the medium include Burkholder (Burk holder).
holder) Minimal medium [Bostian, KL et al., Proc.Natl.Acad.Sc (Proc.Natl.Acad.Sc)
i.USA), 77, 4505 (1980)], or 0.5
SD medium containing% casamino acids [Bitter, GA et al., Procedures of the National Academy.
Of Sciences of the USA (Pro
c.Natl.Acad.Sci.USA), 81, 5330 (198).
4)]. The pH of the medium is preferably adjusted to about 5-8. Culturing is usually performed at about 20 to 35 ° C for about 24 to 7
Perform for 2 hours, add aeration and agitation as needed.

When culturing a transformant whose host is an insect cell or an insect, the medium is Grace's Insect Medi
um (Grace, TCC, Nature), 195,78
8 (1962)) to which an additive such as immobilized 10% bovine serum is appropriately added. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culturing is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science, 122].
Vol. 501 (1952)], DMEM medium [Virology, Volume 8, 396 (1959)], RPM
I1640 medium [The Journal of the American Medical Association (The Journal of t
he American Medeical Association), 199, 51
9 (1967)], 199 medium [Procedure of the Society for the Biological.
Medicine (Proceeding of the Society for the Biol
ogical Medicine), 73, 1 (1950)]. The pH is preferably about 6-8. Culturing is usually carried out at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and agitation are added if necessary. As described above, the antibody protein of the present invention can be produced inside the transformant cells, on the cell membrane or outside the cells.

The target antibody protein of the present invention can be separated and purified from the culture thus obtained, for example, by the following method. When the antibody protein of the present invention is extracted from cultured bacterial cells or cells, the bacterial cells or cells are collected by a known method after culturing, suspended in a suitable buffer solution, and then subjected to ultrasonic waves, lysozyme and / or freeze-thawing. After destroying the bacterial cells or cells by the method, a method of obtaining a crude extract of the antibody protein by centrifugation or filtration is appropriately used. In the buffer, protein denaturants such as urea and guanidine hydrochloride, Triton X-100 ^™
And the like. When the antibody protein is secreted into the culture medium, after the culture is completed, the bacterial cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected. Purification of the thus obtained culture supernatant or the antibody protein contained in the extract can be carried out by appropriately combining separation / purification methods known per se. As these known separation / purification methods, solubility-based methods such as salting-out and solvent precipitation methods, dialysis methods, ultrafiltration methods, gel filtration methods, and SDS-polyacrylamide gel electrophoresis methods are mainly used to determine the molecular weight. Method utilizing difference, method utilizing difference in charge such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography , Etc. can be separated and purified using a method utilizing a difference in isoelectric points such as isoelectric focusing.

The protein of the present invention or a salt thereof can be produced by a method known per se for protein synthesis, or by cleaving the protein of the present invention with an appropriate protease. The protein synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is, the target protein can be produced by condensing the partial peptide or amino acid constituting the protein of the present invention with the remaining portion and removing the protecting group when the purified product has the protecting group. For the known condensation method and removal of the protecting group,
For example, the methods described below may be mentioned.

M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Pu
blishers, New York (1966) Schroeder and Luebke, The peptide,
Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Chemistry of Proteins IV, 205, (1977) Supervised by Haruaki Yajima, Development of Pharmaceuticals Volume 14 Peptide Synthesis Hirokawa Shoten The protein of the present invention can be purified and isolated by a combination of purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the protein obtained by the above method is a free form, it can be converted into an appropriate salt by a known method, and conversely, when it is obtained with a salt, it can be converted into a free form by a known method. it can.

The antibody of the present invention obtained as described above,
The complex, protein (recombinant protein of anti-female hormones antibody) and / or phage can be used as a reagent when quantitatively measuring environmental hormones, or by immobilizing it on various carriers. It can be used for producing an affinity column for concentrating the protein.
Further, the application range of the antibody, complex and / or protein of the present invention is expanded by identifying female hormones that bind (ie, cross-react) to the antibody, complex, protein and / or phage of the present invention. be able to. Furthermore, the present invention provides a kit for measuring or quantifying female hormones and a kit for concentrating female hormones, which comprises the antibody, complex and / or protein of the present invention.

The above kit may contain only one kind of the antibody, complex, protein and / or phage of the present invention, but a plurality of different kinds of the antibody, complex, protein and / or phage of the present invention may be contained. Alternatively, it can include a phage. For example, specific female hormones can be specifically measured and quantified by using a kit containing a plurality of complexes having different cross-reactivity. Specifically, in the case of specifically measuring and quantifying a female hormone having a certain characteristic in a sample containing a plurality of female hormones, the complex that binds to the female hormone and the female hormone are substantially It may be used in combination with a complex that does not bind to (but preferably has similar cross-reactivity to other female hormones contained in the sample). The present invention also provides such a method.

As the method for measuring female hormones using the antibody, complex, protein and / or phage of the present invention, radioisotope immunoassay (RIA method) and ELISA method (En
gvall, E. , Methods in Enzymol. , 70, 419-439
(1980)), fluorescent antibody method, plaque method, spot method, agglutination method, Ouchterlony, and various other methods generally used for antigen detection ("Hybridoma method and monoclonal antibody", published by R & D Planning Co., Ltd. , P. 30-p. 53, March 5, 1982). The ELISA method is widely used from the viewpoint of sensitivity, simplicity, and the like.

The carrier for immobilizing the antibody, complex, protein and / or phage of the present invention is, for example, a microplate (eg, 96-well microplate, 24
Well microplate, 192-well microplate, 384-well microplate, etc.), test tube (eg, glass test tube, plastic test tube), glass particle, polystyrene particle, modified polystyrene particle, polyvinyl particle, latex (eg, polystyrene / latex) ), Nitrocellulose membrane, cyanogen bromide activated filter paper, D
Examples thereof include BM activated filter paper, granular solid phase (eg, sepharose, sephadex, agarose, cellulose, sephacryl, etc.), iron-containing polycarbonate film, magnet-containing beads and the like.

To carry the antibody, complex, protein and / or phage of the present invention on a carrier, a method known per se [eg, the above-mentioned "enzyme immunoassay" Nos. 268-2.
96 pages, "Affinity Chromatography Handbook" (Amersham Pharmacia Biotech Co., Ltd. (published on December 20, 1998)) and the like.

In the immunological concentration method of the present invention, a large amount of sample is passed through the immunoadsorbent column,
By mixing with the immunoadsorbent particles, by utilizing the antigen-antibody reaction, the target female hormone, its degradation product or a mixture thereof is captured by the immunoadsorbent, and then,
Changing pH (lowering the PH2.5～3, like raised to pH 11.5), (such as 1M NaCl) changes in ionic strength, changes in polarity (10% dioxane, 50% ethylene glycol, 3M chaotropic salt (SCN ^-, CC1 ₃
COO ⁻ , I ⁻ ), etc.), a protein denaturing agent (8 M urea, 6 M guanidine hydrochloride, etc.) or by elution by a known method such as dissociation by electrophoresis to obtain a target substance having less immunological impurities. , It can be concentrated at a high magnification of several thousand to tens of thousands times. As a result, female hormones that are present in the environment only in trace amounts, their degradation products, or mixtures thereof are concentrated at a much higher magnification than conventional concentration methods such as solvent extraction and solid phase extraction. In addition, it is possible to obtain a concentrated solution having a low content of contaminants and the like that interfere with the determination.

In the present specification and drawings, when an abbreviation is used for a base or amino acid, IUPAC-IUBC
ommission on Biochemical Nomenclature or abbreviations commonly used in the field,
An example is given below. When an amino acid may have an optical isomer, the L form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid a: A: adenine t: T: thymine g: G: guanine c: C: cytosine x: unidentified RNA: ribonucleic acid mRNA: messenger ribonucleic acid amino acid abbreviation 3 letters: 1 Character: Japanese name Gly: G: Glycine Ala: A: Alanine Val: V: Valine Leu: L: Leucine Ile: I: Isoleucine Ser: S: Serine Thr: T: Threonine Cys: C: Cysteine Met: Glu: M: Melu: M: : E: Glutamic acid Asp: D: Aspartic acid Lys: K: Lysine Arg: R: Arginine His: H: Histidine Phe: F: Phenylalanine Tyr: Y: Tyrosine Trp: W: Tryptophan Pro: P: As: Proline As: Gin Gln: Q: glutamine Asx: B: Asn + Asp Glx: Z: Gln + Glu Xaa: Unknown or other amino acid

[0060]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. The mouse hybridoma cells produced in Example 1 below were deposited under the Budapest Treaty at the Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology, Central 6th 1-1-1, Higashi 1-1-1, Tsukuba, Ibaraki, Japan. The deposit number and deposit date are shown below. mouse-hybridoma E2-73: Deposit number: FERM BP-7569 Deposit date: April 25, 2001 mouse-hybridoma E1-420: Deposit number: FERM BP-7568 Deposit date: April 25, 2001 mouse-hybridoma EE2 -227: Deposit number: FERM BP-7567 Deposit date: April 25, 2001 Also, the Escherichia coli transformant produced in Example 5 below was 1-1, Higashi, Tsukuba, Ibaraki, Japan under the Budapest Treaty. -1 Central 6th National Institute of Advanced Industrial Science and Technology The National Institute of Advanced Industrial Science and Technology has been deposited at the Patent Biological Depository Center, and the deposit numbers and deposit dates are shown below. Escherichia coli TG / pCAN-E2scFvHL: Deposit number: FERM BP-7912 Deposit date: February 22, 2002

Reference Example 1 1. 1. Obtaining monoclonal antibody 1-1 Preparation of hapten (1) Preparation of hapten for ethinyl estradiol antibody 1.0 g of ethinyl estradiol (EE2) and 0.76 g of sodium methylate were dissolved in 35 ml of ethanol, and 0.41 g of monochloroacetic acid was further added. The mixture was heated under reflux for 22 hours, concentrated under reduced pressure, and about 100 ml of water and ethyl acetate were added to separate the layers.
After washing the aqueous layer with 50 ml of ethyl acetate, the aqueous layer was acidified with concentrated hydrochloric acid (pH 1-2). After extraction with 100 ml and 50 ml of ethyl acetate, the organic layer was washed with 30 ml of saturated saline and dried over anhydrous sodium sulfate. A part of the solution was crystallized by allowing the concentrated solution under reduced pressure to stand at -20 ° C for 2 days. A part of this was collected and used as a seed crystal. The concentrated solution was dissolved in a small amount of acetone, hexane and seed crystals were added to precipitate crystals. The target product EE2-3-carboxymethyl ether (EE2-3CME) was obtained by drying the crystals collected by filtration under reduced pressure.

(2) Preparation of hapten for 17β-estradiol antibody 1.0 g of 17β-estradiol (E2) and 0.82 g of sodium methylate were dissolved in 35 ml of ethanol, and 0.45 g of monochloroacetic acid was further added, followed by heating under reflux for 2 days. , After concentration under reduced pressure,
About 300 ml of water and about 200 ml of ethyl acetate were added for liquid separation. The aqueous layer was washed with 50 ml of ethyl acetate and then acidified with concentrated hydrochloric acid.
(pH 1-2). After extracting twice with 100 ml of ethyl acetate, the organic layer was washed with 30 ml of saturated saline and dehydrated with anhydrous sodium sulfate. The concentrated solution under reduced pressure was dissolved in a small amount of acetone, and isoprovir ether (IPE) was added to precipitate crystals. The crystals collected by filtration are washed with IPE and then dried under reduced pressure to obtain the target product E.
2-3-carboxymethyl ether (E2-3CME) was obtained.

(3) Preparation of hapten for estrone antibody Estradiol-3,17-diacetate (E2-3,17-diAce) 2
0 g is dissolved in 25 ml of acetic acid, and 4/5 of a mixed solution of 9 g of chromic anhydride, 27 ml of acetic acid and 4 ml of water is gradually added while cooling. After stirring overnight, water was added and the mixture was extracted with chloroform, washed with potassium carbonate solution, dehydrated with sodium sulfate, and concentrated under reduced pressure. After the concentrated solution was dissolved in benzene, it was subjected to column chromatography (Wako gel 2w) and eluted with hot benzene to collect crude 6-oxo-E2-3,17-diAce (remove the raw material while confirming by TLC). . This substance was dissolved in 2-methoxy-ethanol, 20% NaOH was added in the same amount, and the mixture was reacted at 100 ° C for 1 hour. After acidification with dilute hydrochloric acid, elution with ethyl acetate, dehydration with sodium sulfate, concentration under reduced pressure, and recrystallization from methanol (6oxo-E2). 6oxo-E2 300mg ethanol
Dissolve in 2 ml, add 50 mg of fine powder of potassium carbonate, add 50 μl of benzyl bromide, and stir (70 ℃, 2 hours)
The concentrate was recrystallized from methanol (6-oxo-E2-3-benzyl ether). 6-oxo-E2-3-benzyl ether was dissolved in methanol, and carboxymethoxylamine 1 / 2HCl and sodium methoxide (1.2 molar amount)
Was dissolved in a small amount of water, diluted with methanol and added. The reaction was carried out at room temperature for 1 hour, 2/3 was evaporated, acidified with water and HCl, extracted with ethyl acetate and dried over sodium sulfate. After dissolving in hexane, the product was applied to a silica gel column and eluted with methanol to purify it, and then recrystallized from methanol. 0.6 g of the recrystallized substance is dissolved in 2 ml of acetone,
Add half of the mixture of chromic anhydride 1 g, acetic acid 5 ml, water 1 ml,
After the exotherm had ended, it was extracted with ethyl acetate and evaporated to dryness (6-oxo-E1-3-benzyl ether). 6-oxo-E1-
Dissolve 400 mg of 3-benzyl ether in 5 ml of methanol,
% Pd.C 100 mg was added and hydrogen was stirred. After reacting for 1 hour, the mixture was evaporated to dryness and recrystallized from methanol to obtain the desired product 6-oxo-E1-6 carboxymethyl oxime (E1-6CMO).

1-2 Preparation of Immunogen For each of the three haptens obtained in 1-1, 0.1 mol of hapten, 0.14 mol of water-soluble carbodiimide, and 0.14 mol of N-hydroxysuccinimide were added overnight in 2 ml of dimethyl sulfoxide. Reacted to make an activated ester. Next, 10 mg of keyhole limpet hemocyanin (KLH) was dissolved in a 0.13 molar sodium bicarbonate (NaHCO ₃ ) solution, 200 μl of the activated ester was added, and the mixture was reacted overnight at 4 ° C. Unreacted reagents were removed by dialysis against Dulbecco's phosphate buffer (PBS) and frozen and stored as an immunogen.

1-3 Immunization The immunogen obtained in 1-2 was dissolved in PBS at 500 μg / ml and added to an equal volume of Freund's adjuvant or RIBI adjuvant system. After sufficiently emulsifying, 100 μg / mouse was subcutaneously administered to BALB / C mice (female), and booster immunization was carried out at 2 week (Freund) or 3 week (RIBI) intervals. After 5 to 6 booster immunizations, the same immunogen was intravenously administered (50 μg / 0.1 ml P
(BS / mouse) The final immunization.

1-4 Spleen was excised from the mouse which was finally immunized by cell fusion 1-3 (3 days after the last immunization) to prepare spleen cells. Separately cultured mouse myeloma cells and spleen cells were contacted at a ratio of 1: 5 in the presence of polyethylene glycol (average molecular weight 4000), and cell fusion was performed to obtain fused cells (hybridomas). After suspending this hybridoma in HAT medium,
It was rolled up in a 6-well microplate and cultured in a carbon dioxide gas incubator (37 ° C, 5% CO ₂ ).

1-5 Screening of hybridoma (1) Preparation of assay plate Goat anti-mouse IgAGM antibody (ICN / Cappel, # 55461) was used in PBS.
Dissolve 50 μg / ml in 100 μl / well in a microplate.
Added in. After reacting overnight at 4 ° C, after washing with PBS containing 0.05% Tween20 (T-PBS) (twice at 300 μl / well), Block Ace diluted 4 times with PBS (Snow Brand Milk Products Co., Ltd.), (Tokyo) was added at 200 μl / well. After reacting at 4 ° C overnight or more, it was stored refrigerated until use.

(2) Preparation of antigen-enzyme complex (labeled antigen) For each of the three haptens prepared in 1-1, 0.1 mol of hapten, 0.14 mol of water-soluble carbodiimide, and 0.14 mol of N-hydroxysuccinimide were added to dimethyl sulfoxide. The activated ester was made by reacting in 2 ml overnight. Next, add horseradish peroxidase (HRP) 10 mg to 0.13
It was dissolved in 10 ml of a molar sodium bicarbonate (NaHCO ₃ ) solution, 15 μl of the activated ester was added thereto, and the mixture was reacted overnight at 4 ° C.
The unreacted reagent was removed by ultrafiltration and refrigerated at a concentration of 3 mg / ml.

(3) Primary screening (antigen-binding ability test) Regarding the microplate in which cells were involved in 1-4,
100 μl of the culture solution in the well in which cell growth was confirmed was added to the assay plate prepared in (1) (washed with PBS (or T-PBS) before use (300 μl / well twice)). After reacting for 1 hour at room temperature, wash with T-PBS (300 μl / well 3 times), 1-
The labeled antigen of the target antibody prepared in 5 (2) was diluted 5000-fold with T-PBS and added to the microplate. After reacting at room temperature for 1 hour, the plate was washed with T-PBS (three times at 300 μl / well), and TMB peroxidase substrate kit (Nippon Bio-Rad, Tokyo, # 172-10).
66: The following chromogenic substrate) was added at 100 μl / well. Thirty
After reacting at room temperature for 1 minute, 1N phosphoric acid was added at 100 μl / well,
The color reaction was stopped. The absorbance was read at a wavelength of 450 nm, and cells having an absorbance of more than 1 were scaled up to a 24-well microplate.

(4) Secondary screening (female hormone inhibition test) For cells scaled up to a 24-well microplate in 1-5 (3), a well culture medium in which sufficient cell growth was confirmed was prepared in (1). Assay plate (PBS before use
(Or T-PBS) after washing (300 μl / well twice) to 100 μ
l was added to each well. After reacting for 1 hour at room temperature, TP
Wash with BS (300 μl / well three times) and measure the female hormone (1 ng / ml in 10% methanol (hereinafter MeOH)) to be measured or 10% MeOH alone (control) and the labeled antigen (TP) of the target antibody.
(5000-fold diluted with BS) were mixed in equal amounts and added to the microplate. After reacting at room temperature for 1 hour, wash with T-PBS (300 μl
chromogenic substrate was added at 100 μl / well. Thirty
After the reaction at room temperature for 1 minute, 100 μl / well of 1N phosphoric acid was added to stop the color reaction. The absorbance was read at a wavelength of 450 nm, compared with the control, cloning was carried out according to a conventional method for cells in which the decrease in the absorbance due to the presence of a female hormone was confirmed to be 20% or more, and a target antibody-producing hybridoma strain was obtained as follows. .

(1) Anti-estradiol monoclonal antibody-producing mouse hybridoma cell line E2-73 (FERM BP-7569), (2) Anti-estron monoclonal antibody-producing mouse hybridoma cell line E1-420 (FERM BP-7568), (3) Anti-ethinyl estradiol monoclonal antibody-producing mouse hybridoma cell line EE2-227 (FERM BP-7567), (4) Anti-ethinyl estradiol monoclonal antibody-producing mouse hybridoma cell line EE2-3 (the degree of resistance of the antibody obtained from this strain to detergents is , EE2-277 strain.) (5) Ethinyl estradiol anti-monoclonal antibody-producing mouse hybridoma cell line EE2-8-18 (the degree of resistance of the antibody obtained from this strain to detergents
Slightly lower than that of E2-277 strain. )

Mouse hybridoma cell E2-73 described above
Strain (FERM BP-7569), mouse hybridoma cell E1-420 strain (FERM BP-7568),
Mouse hybridoma cell line EE2-227 (FERM
BP-7567) has been deposited at the Japan Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology from April 25, 2001 with the above-mentioned deposit number.

Example 1 Determination of N-terminal amino acid sequence of anti-estradiol monoclonal antibody Anti-estradiol monoclonal antibody-producing mouse hybridoma cells (E2-73 strain) (FERM BP-7
569) From the culture supernatant, Protein A column (PROSEP-A,
The antibody protein purified using Air Brown Co., Ltd.) was separated and purified into heavy chains and light chains by SDS-polyacrylamide gel electrophoresis, and transferred to a PVDF membrane. After staining with Ponceau S, the protein band was cut out. The proteins on the excised membrane were labeled with Pfu Pyroglutamate Aminopeptida.
se (purchased from Takara Shuzo Co., Ltd.) After treatment with 2 mU at 50 ° C. for 17 hours, the N-terminal amino acid sequence was determined with a gas phase protein sequencer (Model 494 Applied Biosystems Japan), and the following results were obtained. E2-73 heavy chain: VQLQQSGAELARPGASVKLS (SEQ ID NO: 21) E2-73 light chain: DIVMTQSPTFLSTSLGDKIT (SEQ ID NO: 22)

Example 2 Purification of Anti-Estradiol Monoclonal Antibody mRNA Anti-Estradiol Monoclonal Antibody-Producing Mouse Hybridoma Cells (Strain E2-73) (FERM BP-7
569) 1 × 10 ⁷ cells / ml was suspended in 1 ml of an RNA preparation solution (ISOGEN solution, purchased from Wako Pure Chemical Industries) to disrupt the cells, and RNA was extracted from the cells according to the manual. RNA concentration is
The concentration was calculated from the absorbance at 260 nm, and from about 100 μg of the obtained RNA, mRNA purification kit (Oligotex-dT30 <super> mRNA Purific
ation kit, purchased from Takara Shuzo Co., Ltd.), about 1.5 μg
Was obtained.

Example 3 Anti-Estradiol Monoclonal Antibody Obtaining heavy chain and light chain variable region cDNA fragments, determining the nucleotide sequences, and deducing the amino acid sequences of the heavy and light chain variable regions About 100 ng of each mRNA for
irst Strand cDNA Synthesis Kit ReverTra Ace -α ^TM
(Purchased from Toyobo Co., Ltd.) was used. Then each chain cD
The amplification of the NA fragment was performed using the heavy chain primer and the light chain primer shown below, which were devised from the N-terminal amino acid sequence obtained in Example 1, and using Pfu Turbo (purchased from Toyobo Co., Ltd.) enzyme. PCR was performed with a personal cycler (purchased from Biometra) at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 2 minutes, and the number of repetitions was 30 times. E2-73 Heavy chain primer <B: C, G, T> Forward 5'- CTATGCGGCCCAGCCGGCCATGGTBCAGCTGCAGCAGTCTGG -3 '(SEQ ID NO: 23) Back 5'- ACCACTCTCACAGTCTCCTCGG -3' (SEQ ID NO: 24) E2-73 For light chain Primer <Y: C, T> Forward 5'- CTCAGTCTCCAGAYATCGTGATGACYCAGTCT -3 '(SEQ ID NO: 25) Back 5'- GGGACCAAGCTGGAAATAAAACGGA -3' (SEQ ID NO: 26) As a result, the heavy chain and light chain of the anti-estradiol monoclonal antibody A cDNA fragment for the variable region was obtained. Each fragment was purified from 2% agarose after electrophoresis using a micro spin column (purchased from Amersham Pharmacia Biotech Co., Ltd.). Next, the nucleotide sequence of each variable region cDNA was determined by the dideoxy method, and its amino acid sequence was estimated. Use the above-mentioned PCR primers for sequencing, and use Big Dye Terminator Cycle Sequencing.
After performing a reaction with a Personal cycler (purchased from Biometra) using the Ready Reaction Kit (purchased from Applied Biosystems Japan), a DNA sequencer 310
The base sequence was determined by (Applied Biosystems Japan).

Example 4 Anti-Estradiol Monoclonal Antibody Heavy Chain and Light Chain Single chaining of variable region cDNAs (single chain variable site antibody; scFv) and construction of a plasmid containing scFv fragments Obtained heavy chain and light chain Each fragment of the peptide linker ((GG
GGS) X3) (SEQ ID NO: 27), and single-stranded (scFv)
And The scFv DNA fragment was ligated to HincII-cut pUC19 using a ligation kit (purchased from Takara Shuzo), and after the reaction, added to JM109 competent cells (purchased from Toyobo) for transformation. The plasmid was purified from the transformant, and the entire nucleotide sequence of the cDNA insert was determined by the dideoxy method. Then, it was confirmed that they match the nucleotide sequences of the above variable regions (SEQ ID NOS: 1 and 3),
The amino acid sequence was determined (SEQ ID NO: 2 and 4). That is, SEQ ID NO: 1 is mouse hybridoma cell E2-7
The nucleotide sequences of the heavy chain variable region genes of the antibodies produced by the 3 strains are shown, and SEQ ID NO: 2 shows the corresponding amino acid sequence. SEQ ID NO: 3 is mouse hybridoma cell E2-7
The nucleotide sequences of light chain variable region genes of antibodies produced by 3 strains are shown, and SEQ ID NO: 4 shows the corresponding amino acid sequence.

Example 5 Anti Estradiol Monochrome
Expression of null antibody variable region CDNA of anti-estradiol monoclonal antibody variable region
To express the recombinant phage production system.
(Purchased from Amersham Pharmacia Biotech)
I used it. In order to use this system
ScFv and pC excised with restriction enzymes SfiI and NotI
ANTAB 5E Phagemid (Amersham Pharmacia Bio
Quick Ligation Kit (purchased from Tech) (purchased from NEB)
Ligated with the plasmid pCANTAB-5E / E2-
73 scFvHL was obtained. The obtained plasmid was designated as TG1 compete
Cell (E. coli TG-1) (purchased from Toyobo)
Transformed by cloning and plasmid pC
Transformant Escherichi harboring ANTAB-5E / E2-73scFvHL
A coli TG / pCAN-E2scFvHL was produced. In addition, this
The transformant obtained in this manner was used for the helper phage M13KO7.
(Purchased from Amersham Pharmacia Biotech)
Thus, an antibody-expressing phage was obtained. Then enzyme free
Expression scFv on this phage by epidemiology (ELISA)
The reactivity of the antibody with estradiol was confirmed. EL
ISA is the hapten protein complex on the plate) β-Estr
adiol 6- (o-CARBOXY-METHYL) OXIME: Purchased from BSA SIGMA
100 μl) was added and left at 4 ° C. overnight. Then 3
Block with% skim milk for 1.5 hours at 37 ° C.
After washing twice with PBS, the expressed scFv and final concentration 1 mg / ml, 0.
Plate the mixture of 037 mg / ml and 0 mg / ml free antigen
100 μl was added and the mixture was allowed to stand at room temperature for 3 hours. Then 0.5% T
Washed twice with ween / PBS and once with PBS, and diluted 500 times there
HRP / Anti-M13: Second antibody (Amersham Pharmacia by
(Purchased from Oteku Co., Ltd.)
It was Then, wash with 0.5% Tween / PBS twice and PBS once,
Substrate solution (0.2% o-phenylenediamine solution, 0.003% H₂
O ₂) Was added and reacted at room temperature for 20 minutes, then the reaction stop solution (4N H
₂SO_Four) Was added and the absorbance was measured. (492nm) (Figure 1)
From the results obtained, this scFv has a female hormone binding ability.
I was able to confirm.

Example 6 Determination of N-Terminal Amino Acid Sequence of Anti-Estrone Monoclonal Antibody Anti-Estrone Monoclonal Antibody-Producing Mouse Hybridoma Cells (E1-420 Strain) (FERM BP-756
8) The antibody protein purified from the culture supernatant using Protein A column (PROSEP-A, purchased from Air Brown Co., Ltd.) was analyzed by SDS-polyacrylamide gel electrophoresis for the heavy chain,
The light chain was separated and purified, and transferred to a PVDF membrane. Ponceau S
After staining with, the protein band was cut out. Pfu Pyroglutamate Aminopeptidase
(Purchased from Takara Shuzo Co., Ltd.) After treatment with 2 mU at 50 ° C. for 17 hours, the N-terminal amino acid sequence was determined with a gas phase protein sequencer (Model 494 Applied Biosystems Japan), and the following results were obtained. E1-420 heavy chain: IQLVESGPELKKPGETVKIS (SEQ ID NO: 28) E1-420 light chain: DVLMTQTPLTLSVTIGQPA (SEQ ID NO: 29)

Example 7 Purification of Anti-Estrone Monoclonal Antibody mRNA Anti-Estrone Monoclonal Antibody-Producing Mouse Hybridoma Cells (E1-420 Strain) (FERM BP-756
8) 1 × 10 ⁷ cells / ml was suspended in 1 ml of an RNA preparation solution (ISOGEN solution, purchased from Wako Pure Chemical Industries) to disrupt the cells, and RNA was extracted from the cells according to the manual. RNA concentration is 260 nm
Determine the concentration from the absorbance of the
A Purification Kit (Oligotex-dT30 <super> mRNA Purification
kit, purchased from Takara Shuzo Co., Ltd.), using about 1.5 μg of mRNA
Got

Example 8 Anti-Estrone Monoclonal Antibody Heavy chain and light chain variable region cDNA fragments were obtained, their nucleotide sequences were determined, and the deduced cDNAs of the amino acid sequences of the heavy chain and light chain variable regions were synthesized using light chain and heavy chain. About 100 ng of each mRNA for
irst Strand cDNA Synthesis Kit ReverTra Ace -α ^TM
(Purchased from Toyobo Co., Ltd.) was used. Then each chain cD
The amplification of the NA fragment was performed using the heavy chain primer and the light chain primer shown below, which were devised from the N-terminal amino acid sequence obtained in Example 1, and using Pfu Turbo (purchased from Toyobo Co., Ltd.) enzyme. PCR was performed with a personal cycler (purchased from Biometra) at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 2 minutes, and the number of repetitions was 30 times. E1-420 Primer for heavy chain Forward 5'- CTATGCGGCCCAGCCGGCCATGATCCAGTTGGTGCAGTCTGGA-3 '(SEQ ID NO: 30) Back 5'- CGAGGAGACTGTGAGAGTGGT-3' (SEQ ID NO: 31) E1-420 Primer for light chain Forward 5'- CTCAGTCTCCAGATGTTTTGATGACCCAAACT-3 ' (SEQ ID NO: 32) Back 5'- CCGTTTCAGCTCCAGCTTGGTCCC-3 '(SEQ ID NO: 33) This gives the heavy chain of the anti-estron monoclonal antibody,
A cDNA fragment for the variable region of the light chain was obtained. Each fragment is from 2% agarose after electrophoresis and microspin column (purchased from Amersham Pharmacia Biotech Co., Ltd.)
It was purified using. Then, using the dideoxy method,
The nucleotide sequence of each variable region cDNA was determined and its amino acid sequence was estimated. BigDye Terminator Cycle Sequencing Ready
After reaction with Personal cycler (purchased from Biometra) using Reaction Kit (purchased from Applied Biosystems Japan), the nucleotide sequence was determined with DNA sequencer 310 (Applied Biosystems Japan) (SEQ ID NOs: 5 and 7), and amino acid The sequence was determined. (SEQ ID NOS: 6 and 8) That is, SEQ ID NO: 5 is mouse hybridoma cell E1-
CDNA containing the heavy chain variable region of the antibody produced by strain 420
SEQ ID NO: 6 shows the corresponding amino acid sequence. SEQ ID NO: 7 shows the nucleotide sequence of cDNA containing the light chain variable region of the antibody produced by mouse hybridoma cell strain E1-420, and SEQ ID NO: 8 shows the amino acid sequence corresponding thereto.

Example 9 Determination of N-terminal amino acid sequence of anti-ethinyl estradiol monoclonal antibody Anti-ethinyl estradiol monoclonal antibody-producing mouse hybridoma cells (EE2-
3 strains, or EE2-8-18 strain) culture supernatant
The antibody protein purified using in A column (PROSEP-A, purchased from Air Brown Co., Ltd.) was separated and purified into heavy and light chains by SDS-polyacrylamide gel electrophoresis.
Transfer was performed to the DF film. After staining with Ponceau S, the protein band was cut out. Pfu the protein on the excised membrane.
Pyroglutamate Aminopeptidase (purchased from Takara Shuzo Co., Ltd.) was treated with 2 mU at 50 ° C for 17 hours, and then the N-terminal amino acid sequence was determined with a gas phase protein sequencer (Model 494 Applied Biosystems Japan) to obtain the following results. . EE2-3 heavy chain: VTLKESGPGILQPSQTLSLT (SEQ ID NO: 34) EE2-3 light chain: DIVMSQSPSSLAVSVGEKVT (SEQ ID NO: 35) EE2-8-18 heavy chain: VQVKESGPGLVAPSQSL (SEQ ID NO: 36) EE2-8-18 light chain: DIQMTQSPASL ? A? VG? TVT (SEQ ID NO: 3
7) EE2-227 heavy chain: VTLKESGPGILQPSQTL (SEQ ID NO: 38) EE2-227 light chain: DIVMSQSPSSLAVSVGE (SEQ ID NO: 39)

Example 10 Purification of anti-ethinyl estradiol monoclonal antibody mRNA The anti-ethinyl estradiol monoclonal antibody-producing mouse hybridoma cells (EE2-
3 strains, or EE2-8-18 strain) 1 × 10 ⁷ cells / ml,
The cells were disrupted by suspending in 1 ml of an RNA preparation solution (ISOGEN solution, purchased from Wako Pure Chemical Industries), and RNA was extracted from the cells according to the manual. For the concentration of RNA, find the concentration from the absorbance at 260 nm,
From about 100 μg of the obtained RNA, mRNA purification kit (Oligotex-dT
30 <super> mRNA Purification kit, purchased from Takara Shuzo Co., Ltd., was used to obtain about 1.5 μg of mRNA each.

Example 11 Anti-ethynyl estradiol monoclonal antibody Obtaining heavy chain and light chain variable region cDNA fragments and determining the nucleotide sequences, and deducing the amino acid sequences of the heavy chain and light chain variable regions Use about 100 ng of each mRNA for the strand
irst Strand cDNA Synthesis Kit ReverTra Ace -α ^TM
(Purchased from Toyobo Co., Ltd.) was used. Then each chain cD
The amplification of the NA fragment was performed using the heavy chain primer and the light chain primer shown below, which were devised from the N-terminal amino acid sequence obtained in Example 1, and using Pfu Turbo (purchased from Toyobo Co., Ltd.) enzyme. PCR was performed with a personal cycler (purchased from Biometra) at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 2 minutes, and the number of repetitions was 30 times. EE2-3 heavy chain primer <R: A, G> Forward 5'- CTATGCGGCCCAGCCGGCCCAGGTTACTCTRAAARARTC-3 '(SEQ ID NO: 40) Back 5'- CGCAGAGACAGTGACCAGAGT-3' (SEQ ID NO: 41) EE2-3 light chain primer Forward 5'- CTCAGTCTCCAGACATTGTGATGTCACAGTC-3 '(SEQ ID NO: 42) Back 5'- GCGGCCGCCTCATTCCTGTTGAAGCTCTTGAC-3' (SEQ ID NO: 43) EE2-8-18 heavy chain primer Forward 5'- CTATGCGGCCCAGCCGGCCATGGTGCAGGTGAAGGAGTCAGGA-3 '(SEQ ID NO: 44) Back 5'- CGAGGAGACGGTGACTGAGGT-3 '(SEQ ID NO: 45) EE2-8-18 light chain primer <Y: C, TD: A, G, T> Forward 5'- CTCAGTCTCCAGAYATTCAGATGAYDCAGTC-3' (SEQ ID NO: 46) Back 5'- CCGTTTGATTTCCAGCTTGGTGCC-3 '(SEQ ID NO: 47) EE2-227 heavy chain primer <R: A, G> Forward 5'-CTATGCGGCCCAGCCGGCCCAGGTTACTCTRAAARARTC-3' (SEQ ID NO: 40) Back 5'-CGCAGAGACAGTGACCAGAGT-3 '(SEQ ID NO: 41) EE2-8-227 light chain primer Forward 5'-CTCAGTCTCCAGACATTGTGATGTCACAGTC-3' (SEQ ID NO: 41) Number: 48) Back 5'-GCGGCCGCCTCATTCCTGTTGAAGCTCTTGAC-3 '(SEQ ID NO: 49) Thus, the heavy chain of the anti-ethinyl estradiol monoclonal antibody, to give a cDNA fragment for the variable region of the light chain. Each fragment was purified from 2% agarose after electrophoresis using a micro spin column (purchased from Amersham Pharmacia Biotech Co., Ltd.). Then, using the dideoxy method, determine the nucleotide sequence of each variable region cDNA,
Its amino acid sequence was deduced. Use the above PCR primers as the sequencing primers, and use BigDye Terminator Cycle S
After performing a reaction with a personal cycler (purchased from Biometra) using the equencing Ready Reaction Kit (purchased from Applied Biosystems Japan), the nucleotide sequence was determined with a DNA sequencer 310 (Applied Biosystems Japan) (SEQ ID NO: 9, 11, 13 and 1
5), the amino acid sequence was determined. (SEQ ID NO: 10, 1
2, 14 and 16) That is, SEQ ID NO: 9 shows the nucleotide sequence of cDNA containing the heavy chain variable region of the antibody produced by mouse hybridoma cell EE2-3, and SEQ ID NO: 10 shows the corresponding amino acid sequence. . SEQ ID NO: 11 shows the nucleotide sequence of cDNA containing the light chain variable region of the antibody produced by mouse hybridoma cells EE2-3, and SEQ ID NO: 12 shows the corresponding amino acid sequence. SEQ ID NO: 13 is a mouse hybridoma cell EE2-8-18
Shows the nucleotide sequence of a cDNA containing the variable region of the heavy chain of the antibody produced by SEQ ID NO: 14, and SEQ ID NO: 14 shows the corresponding amino acid sequence. SEQ ID NO: 15 shows the nucleotide sequence of cDNA containing the light chain variable region of the antibody produced by mouse hybridoma cell EE2-8-18, and SEQ ID NO: 16 shows the corresponding amino acid sequence. SEQ ID NO: 17 shows the nucleotide sequence of cDNA containing heavy chain variable region of antibody produced by mouse hybridoma cell EE2-227, SEQ ID NO:
18 shows the amino acid sequence corresponding to this. Sequence number:
19 shows the nucleotide sequence of cDNA containing the light chain variable region of the antibody produced by mouse hybridoma cell EE2-227, and SEQ ID NO: 20 shows the corresponding amino acid sequence.

Example 12 Single chain variable region antibody (E1 containing the heavy chain variable region of anti-estron monoclonal antibody and the light chain variable region of anti-estradiol monoclonal antibody)
Preparation of H-E2LscFv) E1 by PCR using the heavy chain variable region (E1H) primers of the anti-estron monoclonal antibody shown below.
H cDNA was amplified. Next, the E2L cDNA was amplified by PCR using the primers for the light chain variable region (E2L) of the anti-estradiol monoclonal antibody shown below.
Heavy chain variable region (E1
PCR using amplified E1H cDNA and E2L cDNA as templates, using forward primer for H) and back primer for light chain variable region (E2L) of anti-estradiol monoclonal antibody
Single-stranded DNA containing E1H cDNA and E2L cDNA (E1H-
E2L cDNA) was prepared. PCR was performed using Pfu Turbo (purchased from Toyobo Co., Ltd.) enzyme at 94 ° C for 30 seconds and 55 ° C for 30 seconds.
Second, 72 ° C., 2 minutes, 30 repetitions. Primer for heavy chain variable region (E1H) of anti-estron monoclonal antibody forward 5'-CTATGCGGCCCAGCCGGCCATGATCCAGTTGGTGCAGTCTGGA-3 '(SEQ ID NO: 30) back 5'-GAGCCACCTCCGCCTGAACCGCCTCCACCCGAGGAGACTGT-3' (SEQ ID NO: 50) light chain of anti-estradiol monoclonal antibody Variable region (E2L) primer forward 5'-CGGAGGTGGCTCTGGCGGTGGCGGATCGGATATCGTGATG -3 '(SEQ ID NO: 51) back 5'-GCGCACCTGCGGCCGCCCGTTTTATTTC -3' (SEQ ID NO: 52)

Example 13 Preparation of single chain variable region antibody (E2H-E1L scFv) containing heavy chain variable region of anti-estradiol monoclonal antibody and light chain variable region of anti-estron monoclonal antibody Anti-estradiol monoclonal antibody shown below The E2H cDNA was amplified by PCR using the primers for the heavy chain variable region (E2H) of. Then, the E1L cDNA was amplified by PCR using the primers for the light chain variable region (E1L) of the anti-estron monoclonal antibody shown below. Using forward primer for heavy chain variable region (E2H) of anti-estradiol monoclonal antibody and back primer for light chain variable region (E1L) of anti-estron monoclonal antibody, amplified E2H cDNA and E1L cDNA were used as templates
Single-stranded DNA containing E2H cDNA and E1L cDNA by PCR
(E2H-E1L cDNA) was prepared. In addition, PCR is Pfu Turbo
(Purchased from Toyobo Co., Ltd.) Using enzyme, 94 ℃ for 30 seconds, 55
C. 30 seconds, 72.degree. C. 2 minutes, repeated 30 times. Primer for heavy chain variable region (E2H) of anti-oestradiol monoclonal antibody forward 5'-CTATGCGGCCCAGCCGGCCATGGTBCAGCTGCAGCAGTCTGG-3 '(SEQ ID NO: 23) back 5'-GAGCCACCTCCGCCTGAACCGCCTCCACCCGAGGAGACTGT-3' (SEQ ID NO: 53) light chain of anti-estron monoclonal antibody Variable region (E1L) primer forward 5'-CGGAGGTGGCTCTGGCGGTGGCGGATCGGATGTTTTGATG -3 '(SEQ ID NO: 54) back 5'-GCGCACCTGCGGCCGCCCGTTTCAGCTC-3' (SEQ ID NO: 55)

Example 14 Single chain variable region antibody (E1H-E2
Preparation of L scFv and E2H-E1L scFv) Expression Vector The single-stranded DNA (E1H-E2L cDNA) obtained in Example 13 was digested with restriction enzymes SfiI and NotI. Then E1H-E2L cD
PCANTAB5E pre-digested with 41 ng NA and SfiI and NotI
(Purchased from Amersham Pharmacia Biotech) 90ng
DNA ligation kit (TaKaR
Solution A (Reaction Buffer) 50 μl and solution B (Enzyme So
lution) 10 μl was added, and the mixture was reacted at 16 ° C. for 2 hours. Then, the DNA sample was purified by alkaline dissolution and ethanol precipitation and dissolved in sterile ultrapure water. Then obtained
2 μl of DNA sample was added to TG-1 competent cell (STRATAGENE
Purchased from) and mixed with GENE Pulser II Electroportion
It was electroporated by System (BIO RAD) (200Ω, 2.5KV, Capa 25). Then add the appropriate amount of 2 to this solution.
× YTG (1 × YTG: Bacto-tryptone 8.5 in 1000 ml of sterilized water)
g, Bacto-yeast extract 5g, NaCl 2.5g, 1% Glucose)
Was added to the tube, and the mixture was transferred to a tube and cultured with shaking at 37 ° C. for 1 hour, then plated on a medium plate and cultured overnight at 37 ° C. The next day, after picking up the colonies on the plate and culturing overnight in LB medium, the phagemid was purified by alkaline lysis and ethanol precipitation, and it was confirmed that the target insert DNA was inserted into the phagemid by SfiI and NotI treatment. Single chain variable region antibody (E1H-E2L scFv) expression vector pCANTAB5E / E1
H-E2L scFv was obtained. In addition, E2H-E1L cDNA was treated by the same method as E1H-E2L cDNA, and single chain variable region antibody (E2H-E1L scFv) expression vector pCANTAB5E / E2H-E1L scFv
Got

Example 15 Single Chain Variable Region Antibody (E1H-E2
L scFv and E2H-E1L scFv) measurement of the binding ability to estrone and estradiol of the expression phage E1H-E2L scFv colonies containing E2H-E1L scFv expressing phage were picked up from the medium plate, respectively.
1.6 ml of LB medium containing ampicillin (concentration 100 μg / ml) was inoculated and cultured with shaking at 37 ° C. overnight. Next day, the obtained culture solution
200 μl was added to 20 ml of LB medium containing ampicillin (concentration 100 μg / ml), and the mixture was shake-cultured at 37 ° C. for 2 hours. Two hours later,
Add 20 μl of helper phage M13KO7 solution to this medium and
Culturing was performed for 1 hour while still standing at ℃. One hour later, kanamycin (final concentration 50 μg / ml) was added to this medium, and the mixture was cultured at 28 ° C. with shaking overnight. Then, the culture solution was transferred to a centrifuge tube and centrifuged (9500 rpm, 4 ° C., 35 minutes). Transfer the supernatant (containing phage) to another centrifuge tube and centrifuge (11000rp
m, 4 ° C, 30 minutes). The supernatant (containing phage) was transferred to another centrifuge tube, mixed with 5 ml of 20% PEG6000 and 2.5M NaCl, and left standing on ice for 1 hour. After 1 hour, centrifuge (11000rp
m, 4 ° C., 30 minutes), the supernatant was discarded and centrifuged briefly, and then the supernatant was completely removed. The obtained precipitate was dissolved in 300 μl of PBS and stored at 4 ° C. Then, in the same manner as described above, the immunogens E1-6CMO: Ova (hereinafter abbreviated as E1-Ova, Ova represents ovalbumin) and E2-3CME: Ova (hereinafter E2-Ova).
Was omitted) was prepared. Then Ova, E1-Ova or E2-Ova
(10 μg / ml each) 100 μl was added to each well and left overnight at 4 ° C. for immobilization. After removing the liquid from the wells, add 300 μl of 3% skim milk to each well and incubate at 37 ° C.
It was left for 1.5 hours. After 1.5 hours, the liquid was removed from the wells and the wells were washed twice with PBS. The solution containing the E1H-E2L scFv expressing phage was diluted 2-fold with PBS, 100 μl of the 2-fold diluted phage solution was added to each well, and the mixture was allowed to stand at room temperature for 2.5 hours. After 2.5 hours, remove the liquid from each well and add 0.05
Wells were washed twice with% Tween / PBS and once with PBS. Then, 100 μl of the second antibody solution (HRP / Anti-M13 (purchased from Amersham Pharmacia) diluted 1: 250 with 0.1% skim milk / PBS) was added to each well, and left at room temperature for 1.5 hours. 1.5 hours later, 0.05% Tween / PBS twice, PBS 1
The wells were washed once. TMB solution (purchased from Wako Pure Chemical Industries, Ltd.) was added and reacted for 30 minutes while shaking. 30 minutes later,
The reaction was stopped by adding 1N HCl to the wells. Then 4
Absorbance at 50 nm was measured. The results are shown in Figure 2. As a result, the absorbance in the well to which the antigen E1-Ova or E2-Ova was immobilized was higher than that in the well to which Ova was immobilized.
From the above, E1H-E2L scFv expressing phage and E2H-E1L scFv
It was revealed that the expressing phage bound to both estrone and estradiol.

Example 16 Single chain variable region antibody (E2H-E1
Change in binding ability to estrone and estradiol by dilution of L scFv) expressing phage By the same method as in Example 15, binding ability of E2H-E1L scFv expressing phage to estrone and estradiol was examined. The phage solution diluted twice was used, but this time, it was diluted 16 times, 64 times, 25 times.
A 6-fold diluted phage solution was used. The results are shown in Figure 3. As a result, the E2H-E1L scFv-expressing phage reacted with the antigens E1-Ova and E2-Ova in a concentration-dependent manner, and Ov at all dilutions.
The absorbance was higher with E1-Ova and E2-Ova than with a. In addition, the antibody strongly reacted with E2-Ova and further with E1-Ova. From the above, it was clarified that E2H-E1L scFv was expressed on the phage and reacted with both antigens.

Example 17 Comparison of binding ability to estrone and estradiol between monoclonal antibody and scFv-expressing phage. Example 5
(Prepared by the same method as above) and monoclonal antibody E1-420
Between the anti-estradiol monoclonal antibody E2-733
The binding ability to the antigen was compared between the scFv-expressing phage (produced in Example 5) in which the heavy chain and light chain variable regions of E. coli were produced and the anti-estradiol monoclonal antibody E2-733. The results are shown in Fig. 4. As a result, all scFv-expressing phages showed the same binding ability to the antigen as the original monoclonal antibody. The reason why the dilution curve of the scFv-expressing phage has a gradual slope is considered to be because the reaction time is short and the reaction does not completely reach equilibrium. In particular, since the phage has a large molecular weight, it is considered that a marked difference appeared in E1 having weak binding. From the above results, a phage antibody useful for detecting female hormones could be obtained.

[0090]

INDUSTRIAL APPLICABILITY The present invention provides genes encoding the heavy and light chains of anti-female hormone antibodies, and their amino acid sequences and base sequences have been clarified. It becomes possible to genetically modify the gene provided by the present invention, in particular, the genes encoding the variable regions of the heavy chain and the light chain, and by expressing the modified gene in a host cell, measurement of female hormones can be performed. It became possible to obtain a large amount of a protein having a more preferable property for binding and quantifying female hormones for quantification and concentration. By using a recombinant microbial cell having this modified antibody gene,
It has become possible to efficiently produce a recombinant protein. As a result, it becomes possible to produce an enzyme immunoassay kit or an antibody affinity column with higher performance at a lower cost. Further, the present invention made it possible to produce a phage antibody that expresses a single-chain antibody having variable regions of heavy and light chains of an anti-female hormone antibody.
As a result, it became possible to produce an antibody suitable for differentiating various estrogens or performing simultaneous analysis by combining these variable region fragments.

[0091]

[Sequence list]                             SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> A protin binding to female hormones and a production thereof <130> A5873 <150> JP 2002-055669 <151> 2002-03-01 <160> 59 <210> 1 <211> 351 <212> DNA <213> Mouse <400> 1 gtgcagctgc agcagtctgg ggctgaactg gctagacctg gggcttcagt gaaattgtcc 60 tgcaaggctt ctgactacac ctttactaac tactggatgc agtggataaa acagaggcct 120 ggacagggtc tggaatggat tgggactatt tctcttggaa atggtgatac cagatacact 180 cagaagttca aggacaaggc cacattgact gcagataaat cctccagtac agtctacatg 240 caactcacca acttggcatc tgaggattct gcggtctatt actgttcaat ctattccttc 300 gttaggggcc ccggaggttg gggccaaggc accactctca cagtctcctc g 351

[0092] <210> 2 <211> 117 <212> PRT <213> Mouse <400> 2 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser 1 5 10 15 Val Lys Leu Ser Cys Lys Ala Ser Asp Tyr Thr Phe Thr Asn Tyr Trp             20 25 30 Met Gln Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly         35 40 45 Thr Ile Ser Leu Gly Asn Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys     50 55 60 Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr Met 65 70 75 80 Gln Leu Thr Asn Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ser                 85 90 95 Ile Tyr Ser Phe Val Arg Gly Pro Gly Gly Trp Gly Gln Gly Thr Thr             100 105 110 Leu Thr Val Ser Ser         115 117

[0093] <210> 3 <211> 324 <212> DNA <213> Mouse <400> 3 gatatcgtga tgactcagtc tcccacattc ttgtctacat cattaggaga caaaatcacc 60 atcacctgca aggccagtca ggatgtgggt cccgctatag cctggtatca acagacacca 120 ggacaatctc ctagactact gatttattgg acatccaccc ggcacactgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctct ccattggcga tgttcagtct 240 gaagacttga cagattattt ctgtcaccaa tatcgcagct atccatacac attcgggggg 300 gggaccaagc tggaaataaa acgg 324

[0094] <210> 4 <211> 108 <212> PRT <213> Mouse <400> 4 Asp Ile Val Met Thr Gln Ser Pro Thr Phe Leu Ser Thr Ser Leu Gly 1 5 10 15 Asp Lys Ile Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Pro Ala             20 25 30 Ile Ala Trp Tyr Gln Gln Thr Pro Gly Gln Ser Pro Arg Leu Leu Ile         35 40 45 Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Gly Asp Val Gln Ser 65 70 75 80 Glu Asp Leu Thr Asp Tyr Phe Cys His Gln Tyr Arg Ser Tyr Pro Tyr                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg             100 105 108

[0095] <210> 5 <211> 354 <212> DNA <213> Mouse <400> 5 atccagttgg tgcagtctgg acctgaactg aagaagcctg gagagacagt caagatctcc 60 tgcaaggctt ctgggtttac cttcagaaac tatggaatga actgggtgag gcagtctcca 120 ggagagggtt taaagtggat gggctggata aacacctaca ctggagagcc aacatatcct 180 gatgccttca agggacggtt tgccatctct ctggaaacct ctgccagcac tgcctatttg 240 cagatcaaca gcctcaaaaa tgaggacacg gctacatatt tctgtgcaag aggggactat 300 aggtaccgtt cctttgacta ctggggccaa ggcaccactc tcacagtctc ctcg 354

[0096] <210> 6 <211> 118 <212> PRT <213> Mouse <400> 6 Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr 1 5 10 15 Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Arg Asn Tyr Gly             20 25 30 Met Asn Trp Val Arg Gln Ser Pro Gly Glu Gly Leu Lys Trp Met Gly         35 40 45 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Pro Asp Ala Phe Lys     50 55 60 Gly Arg Phe Ala Ile Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu 65 70 75 80 Gln Ile Asn Ser Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala                 85 90 95 Arg Gly Asp Tyr Arg Tyr Arg Ser Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Thr Leu Thr Val Ser Ser         115 118

[0097] <210> 7 <211> 339 <212> DNA <213> Mouse <400> 7 gatgttttga tgacccaaac tccactcact ttgtcggtta ccattggaca accagcctcc 60 atctcttgca agtcaagtca gagcctctta aatagtgatg gaaagacata tttgcattgg 120 ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180 tctggagtcc ctgacaggtt ctctggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tttgggagtt tattattgct ggcaagttac acattttcct 300 ctcacgttcg gtgctgggac caagctggag ctgaaacgg 339

[0098] <210> 8 <211> 113 <212> PRT <213> Mouse <400> 8 Asp Val Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser             20 25 30 Asp Gly Lys Thr Tyr Leu His Trp Leu Leu Gln Arg Pro Gly Gln Ser         35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Val                 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys             100 105 110 Arg 113

[0099] <210> 9 <211> 354 <212> DNA <213> Mouse <400> 9 gttactctga aaxagtctgg ccctgggata ttgcagccct cccagaccct cagtctgact 60 tgttctttct ctgggttttc actgagcact tctgatctgg gtgtgagttg gattcgtcag 120 acttcaggaa agggtctgga gtggctggca cacatttact ggaatgatga caaatattat 180 aatccatccc tgaagagccg gctcacaatc tccaaggata cctccggaaa cctggtattc 240 ctcaggatca ccagtgtgga cactgacgat tctgccacat actattgtgc tcgaagtcct 300 ctctacggta gtcttactta ctggggccaa gggactctgg tcactgtctc tgcg 354

[0100] <210> 10 <211> 118 <212> PRT <213> Mouse <400> 10 Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln Thr 1 5 10 15 Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser Asp             20 25 30 Leu Gly Val Ser Trp Ile Arg Gln Thr Ser Gly Lys Gly Leu Glu Trp         35 40 45 Leu Ala His Ile Tyr Trp Asn Asp Asp Lys Tyr Tyr Asn Pro Ser Leu     50 55 60 Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Gly Asn Leu Val Phe 65 70 75 80 Leu Arg Ile Thr Ser Val Asp Thr Asp Asp Ser Ala Thr Tyr Tyr Cys                 85 90 95 Ala Arg Ser Pro Leu Tyr Gly Ser Leu Thr Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ala         115 118

[0101] <210> 11 <211> 654 <212> DNA <213> Mouse <400> 11 gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagtaggaga gaaggtcact 60 ttgaactgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ctacttggct 120 tggtaccagc agaaaccagg acagtctcct aaactgctga tctcttgggc atccactagg 180 gaatctgggg tccctgatcg tttcacaggc agtggatctg ggacagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca gtttattact gcaatcaatt ttattatcta 300 ctcacgttcg gtgctgggac caagctggag ctgaaacggg ctgatgctgc accaactgta 360 tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 420 ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga 480 caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg 540 agcagcaccc tcacgttgac caaggacgag tatgaacgac ataacagcta tacctgtgag 600 gccactcaca agacatcaac ttcacccatt gtcaagagct tcaacaggaa tgag 654

[0102] <210> 12 <211> 218 <212> PRT <213> Mouse <400> 12 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Leu Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser             20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Ser Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Ser Gly Val     50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Asn Gln                 85 90 95 Phe Tyr Tyr Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys             100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu         115 120 125 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe     130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser                 165 170 175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu             180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser         195 200 205 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu     210 215 218

[0103] <210> 13 <211> 355 <212> DNA <213> Mouse <400> 13 gtgcaggtga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcagt 60 tgcactgtct ctgggtttcc attaaccagt tatggtgtac agtgggttcg ccagcctcca 120 ggaaagggtc tggagtggct gggagtaata tggcctggtg gaagcacaaa ttataattcg 180 gctctcatgt ccagactgag catcagcaaa gacaactcca agagccaagt tttcttaaaa 240 atgaacagtc tgcaaactga tgacacagcc atgtactact gtgccggatg gttccaaacg 300 ggaccgaggc tatttgctta ctggggccaa gggactctgg tcactgtctc tgcga 355

[0104] <210> 14 <211> 118 <212> PRT <213> Mouse <400> 14 Val Gln Val Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser 1 5 10 15 Leu Ser Ile Ser Cys Thr Val Ser Gly Phe Pro Leu Thr Ser Tyr Gly             20 25 30 Val Gln Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly         35 40 45 Val Ile Trp Pro Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser     50 55 60 Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys 65 70 75 80 Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala Gly                 85 90 95 Trp Phe Gln Thr Gly Pro Arg Leu Phe Ala Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ala         115 118

[0105] <210> 15 <211> 321 <212> DNA <213> Mouse <400> 15 xxxxxxxxxx xxxxxxxxxx xccagcctcc ctatctgcat ctgtgggaga aactgtcacc 6 0 atcacatgtc gagcaagtga gaatattcac agttatttag catggtatca gcagaaacag 12 0 ggaaaatctc ctcaggtcct ggtctataat gcaaaaacct taggagaagg tgtgtcatca 18 0 aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 24 0 gaagattttg ggacttatta ctgtcaaaat cattatggta gtccgtggac gttcggtgga 30 0 ggcaccaagc tggaatcaaa a 321

[0106] <210> 16 <211> 107 <212> PRT <213> Mouse <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile His Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Val Leu Val         35 40 45 Tyr Asn Ala Lys Thr Leu Gly Glu Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln Asn His Tyr Gly Ser Pro Trp                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ser Lys             100 105 107

[0107] <210> 17 <211> 355 <212> DNA <213> Mouse <400> 17 gttactctga aagagtctgg ccctgggata ttgcagccct cccagaccct cagtctgact 60 tgttctttct ctgggttttc actgagcact tctgatctgg gtgtgagttg gattcgtcag 120 cgttcaggaa agggtctgga gtggctggca cacatttact ggaatgatga caaatactat 180 aatccatacc tgaggagccg gctcacaatc tccaaggata ccgccggaaa cctggtattc 240 ctcaggatca ccagtgtgga cactgacgat tctgccacat actattgtgc tcgaagccct 300 ctctacggta gtctttctta ctggggccaa gggactctgg tcactgtctc tgcga 355

[0108] <210> 18 <211> 118 <212> PRT <213> Mouse <400> 18 Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln Thr 1 5 10 15 Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser Asp             20 25 30 Leu Gly Val Ser Trp Ile Arg Gln Arg Ser Gly Lys Gly Leu Glu Trp         35 40 45 Leu Ala His Ile Tyr Trp Asn Asp Asp Lys Tyr Tyr Asn Pro Tyr Leu     50 55 60 Arg Ser Arg Leu Thr Ile Ser Lys Asp Thr Ala Gly Asn Leu Val Phe 65 70 75 80 Leu Arg Ile Thr Ser Val Asp Thr Asp Asp Ser Ala Thr Tyr Tyr Cys                 85 90 95 Ala Arg Ser Pro Leu Tyr Gly Ser Leu Ser Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ala         115

[0109] <210> 19 <211> 626 <212> DNA <213> Mouse <400> 19 gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagtaggaga gaaggtcact 60 atgaactgca aagccagtca gagtctgctc aacagtagaa cccgaaagaa ccacttggct 120 tggtaccagc agaaaccagg acagtctcca acactgttga tctcttgggc atccactagg 180 gaatctgggg tccctgatcg tttcacaggc agtggatctg ggacagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca gtttattact gcaatcaatt tttttatctg 300 ctcacgttcg gtgctgggac caagctggag ctgaaacggg ctgatgctgc accaactgta 360 tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 420 ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga 480 caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg 540 agcagcaccc tcacgttgac caaggacgag tatgaacgac ataacagcta tacctgtgag 600 gccactcaca agacatcaac ttcacc 626

[0110] <210> 20 <211> 208 <212> PRT <213> Mouse <400> 20 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Asn Cys Lys Ala Ser Gln Ser Leu Leu Asn Ser             20 25 30 Arg Thr Arg Lys Asn His Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Ser Pro Thr Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Ser Gly Val     50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Asn Gln                 85 90 95 Phe Phe Tyr Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys             100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu         115 120 125 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe     130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser                 165 170 175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu             180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser         195 200 205

[0111] <210> 21 <211> 20 <212> PRT <213> Mouse <400> 21 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser 1 5 10 15 Val Lys Leu Ser             20

[0112] <210> 22 <211> 20 <212> PRT <213> Mouse <400> 22 Asp Ile Val Met Thr Gln Ser Pro Thr Phe Leu Ser Thr Ser Leu Gly 1 5 10 15 Asp Lys Ile Thr             20

[0113] <210> 23 <211> 42 <212> DNA <213> Artificial <220> <223> Primer <400> 23 ctatgcggcc cagccggcca tggtbcagct gcagcagtct gg 42

[0114] <210> 24 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 24 accactctca cagtctcctc gg 22

[0115] <210> 25 <211> 32 <212> DNA <213> Artificial <220> <223> Primer <400> 25 ctcagtctcc agayatcgtg atgacycagt ct 32

[0116] <210> 26 <211> 25 <212> DNA <213> Artificial <220> <223> Primer <400> 26 gggaccaagc tggaaataaa acgga 25

[0117] <210> 27 <211> 25 <212> PRT <213> Artificial <220> <223> Linker <400> 27 Gly Gly Gly Gly Ser 1 5

[0118] <210> 28 <211> 20 <212> PRT <213> Mouse <400> 28 Ile Gln Leu Val Glu Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr 1 5 10 15 Val Lys Ile Ser             20

[0119] <210> 29 <211> 19 <212> PRT <213> Mouse <400> 29 Asp Val Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala

[0120] <210> 30 <211> 43 <212> DNA <213> Artificial <220> <223> Primer <400> 30 ctatgcggcc cagccggcca tgatccagtt ggtgcagtct gga 43

[0121] <210> 31 <211> 21 <212> DNA <213> Artificial <220> <223> Primer <400> 31 cgaggagact gtgagagtgg t 21

[0122] <210> 32 <211> 32 <212> DNA <213> Artificial <220> <223> Primer <400> 32 ctcagtctcc agatgttttg atgacccaaa ct 32

[0123] <210> 33 <211> 24 <212> DNA <213> Artificial <220> <223> Primer <400> 33 ccgtttcagc tccagcttgg tccc 24

[0124] <210> 34 <211> 20 <212> PRT <213> Mouse <400> 34 Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln Thr 1 5 10 15 Leu Ser Leu Thr             20

[0125] <210> 35 <211> 20 <212> PRT <213> Mouse <400> 35 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr             20

[0126] <210> 36 <211> 18 <212> PRT <213> Mouse <400> 36 Val Gln Val Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser 1 5 10 15 Leu

[0127] <210> 37 <211> 17 <212> PRT <213> Mouse <400> 37 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Xaa Ala Xaa Val Gly Xaa Thr 1 5 10 15 Val Thr     20

[0128] <210> 38 <211> 17 <212> PRT <213> Mouse <400> 38 Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln Thr 1 5 10 15 Leu

[0129] <210> 39 <211> 17 <212> PRT <213> Mouse <400> 39 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu

[0130] <210> 40 <211> 39 <212> DNA <213> Artificial <220> <223> Primer <400> 40 ctatgcggcc cagccggccc aggttactct raaarartc 39

[0131] <210> 41 <211> 21 <212> DNA <213> Artificial <220> <223> Primer <400> 41 cgcagagaca gtgaccagag t 21

[0132] <210> 42 <211> 31 <212> DNA <213> Artificial <220> <223> Primer <400> 42 ctcagtctcc agacattgtg atgtcacagt c 31

[0133] <210> 43 <211> 32 <212> DNA <213> Artificial <220> <223> Primer <400> 43 gcggccgcct cattcctgtt gaagctcttg ac 32

[0134] <210> 44 <211> 43 <212> DNA <213> Artificial <220> <223> Primer <400> 44 ctatgcggcc cagccggcca tggtgcaggt gaaggagtca gga 43

[0135] <210> 45 <211> 21 <212> DNA <213> Artificial <220> <223> Primer <400> 45 cgaggagacg gtgactgagg t 21

[0136] <210> 46 <211> 31 <212> DNA <213> Artificial <220> <223> Primer <400> 46 ctcagtctcc agayattcag atgaydcagt c 31

[0137] <210> 47 <211> 24 <212> DNA <213> Artificial <220> <223> Primer <400> 47 ccgtttgatt tccagcttgg tgcc 24

[0138] <210> 48 <211> 31 <212> DNA <213> Artificial <220> <223> Primer <400> 48 ctcagtctcc agacattgtg atgtcacagt c 31

[0139] <210> 49 <211> 32 <212> DNA <213> Artificial <220> <223> Primer <400> 49 gcggccgcct cattcctgtt gaagctcttg ac 32

[0140] <210> 50 <211> 41 <212> DNA <213> Artificial <220> <223> Primer <400> 50 gagccacctc cgcctgaacc gcctccaccc gaggagactg t 41

[0141] <210> 51 <211> 40 <212> DNA <213> Artificial <220> <223> Primer <400> 51 cggaggtggc tctggcggtg gcggatcgga tatcgtgatg 40

[0142] <210> 52 <211> 28 <212> DNA <213> Artificial <220> <223> Primer <400> 52 gcgcacctgc ggccgcccgt tttatttc 28

[0143] <210> 53 <211> 41 <212> DNA <213> Artificial <220> <223> Primer <400> 53 gagccacctc cgcctgaacc gcctccaccc gaggagactg t 41

[0144] <210> 54 <211> 40 <212> DNA <213> Artificial <220> <223> Primer <400> 54 cggaggtggc tctggcggtg gcggatcgga tgttttgatg 40

[0145] <210> 55 <211> 40 <212> DNA <213> Artificial <220> <223> Primer <400> 55 cggaggtggc tctggcggtg gcggatcgga tgttttgatg 40

[0146] <210> 56 <211> 14 <212> PRT <213> Artificial <220> <223> Linker <400> 56 Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly 1 5 10

[0147] <210> 57 <211> 18 <212> PRT <213> Artificial <220> <223> Linker <400> 57 Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Ser Gly Ser Thr 1 5 10 15 Lys Gly

[0148] <210> 58 <211> 12 <212> PRT <213> Artificial <220> <223> Linker <400> 58 Gly Ser Thr Ser Gly Lys Pro Ser Glu Gly Lys Gly 1 5 10

[0149] <210> 59 <211> 18 <212> PRT <213> Artificial <220> <223> Linker <400> 59 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly

[Brief description of drawings]

FIG. 1 is a view showing that the estradiol single chain variable region antibody constructed in the present invention binds to 17β-estradiol.

FIG. 2 is a single chain variable region antibody (E1H-E2 constructed according to the present invention.
It is the drawing which confirmed that L scFv, E2H-E1L scFv) binds to estrone and 17β-estradiol. White bars correspond to E1H-E2L scFv, and black bars correspond to E2H-E1L scFv. E1-Ova indicates a complex of estrone and ovalbumin, E2-Ova indicates a complex of 17β-estradiol and ovalbumin, and Ova indicates that ovalbumin is an antigen.

FIG. 3 is a single-chain variable region antibody (E2H-E1 constructed according to the present invention.
It is the figure which confirmed the change of the binding ability with respect to estrone and 17 (beta) -estradiol by the dilution of the phage which expresses LscFv). E1-Ova indicates a complex of estrone and ovalbumin, E2-Ova indicates a complex of 17β-estradiol and ovalbumin, and Ova indicates that ovalbumin is an antigen.

FIG. 4 is a view showing the ability of the single-chain variable region antibody constructed according to the present invention and the original monoclonal antibody to bind to an antigen. FIG. 4 (A) shows the binding ability of estrone single chain variable region antibody and monoclonal antibody E1-420 to estrone. FIG. 4 (B) shows the binding ability of estradiol single-chain variable region antibody and anti-estradiol monoclonal antibody E2-733 to 17β-estradiol. The vertical axis of FIGS. 4A and 4B (% inhibition)
Represents the degree of inhibition of antibody binding to the antigen on the plate by the free antigen by subtracting 100 from the absorbance at each concentration with the absorbance in the absence of free antigen as 100.

Front page continuation (51) Int.Cl. ⁷ identification code FI theme code (reference) C12N 1/21 C12N 7/00 5/10 C12P 21/02 C 7/00 C12Q 1/70 C12P 21/02 G01N 33 / 53 F C12Q 1/70 C12N 15/00 ZNAA G01N 33/53 5/00 A (72) Inventor Yasuhiro Gota 2-1785 Jusohonmachi, Yodogawa-ku, Osaka-shi, Osaka Takeda Pharmaceutical Co., Ltd. Living Environment Company Inner F-term (reference) 4B024 AA11 BA43 CA04 CA05 CA06 CA07 CA09 CA12 DA02 EA03 EA04 GA05 GA11 GA18 HA03 HA08 HA14 4B063 QA01 QA18 QQ75 QQ94 QR41 QR48 QR79 QR82 QS12 QS38 QX01 4B064 AG26 CA10 CA19 CA25 CA01 A049A90A24A90 4A0 4A0 4A0 4A0 4A0 CA46 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA40 DA76 EA50 FA72 FA74

Claims (20)

[Claims] 1. A protein or salt thereof of [a] or [b] below: [a] the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 6, the amino acid represented by SEQ ID NO: 10 Sequence, amino acid sequence represented by SEQ ID NO: 14, amino acid sequence represented by SEQ ID NO: 18,
Or a protein having an amino acid sequence substantially the same as these; [b] the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence represented by SEQ ID NO: 12, the SEQ ID NO: A protein having the amino acid sequence represented by 16, the amino acid sequence represented by SEQ ID NO: 20, or an amino acid sequence substantially the same as these. 2. The following [a ¹ ] to [a ³ ] and [b ¹ ] to
Any of the proteins of [b ³ ] or salts thereof: [a ¹ ] consisting of the amino acid sequence of any of (i) to (v) below, wherein the specific regions are: complementarity determining region 1, complementarity determining region 2, A protein characterized by being selected from the group consisting of complementarity determining region 3, framework region 1, framework region 2, framework region 3, and framework region 4, (i) in SEQ ID NO: 2 1 of the amino acid sequences shown
The amino acid sequence corresponding to the above specific region is SEQ ID NO:
One or more identifications of the same type contained in the amino acid sequence represented by 6, the amino acid sequence represented by SEQ ID NO: 10, the amino acid sequence represented by SEQ ID NO: 14 or the amino acid sequence represented by SEQ ID NO: 18. An amino acid sequence exchanged with an amino acid sequence corresponding to the region, (ii) SEQ ID NO:
In the amino acid sequence represented by 6, the amino acid sequence corresponding to one or more specific regions is represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 10, and the amino acid sequence represented by SEQ ID NO: 14. Represented by SEQ ID NO: 10, or an amino acid sequence exchanged with an amino acid sequence corresponding to one or more specific regions of the same kind contained in the amino acid sequence represented by SEQ ID NO: 18 or (iii) SEQ ID NO: 10. The amino acid sequence corresponding to one or more specific regions in the amino acid sequence is represented by SEQ ID NO: 2,
The amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 14
An amino acid sequence represented by SEQ ID NO: 18 or an amino acid sequence replaced with an amino acid sequence corresponding to one or more specific regions of the same type contained in the amino acid sequence represented by SEQ ID NO: 18, (iv) represented by SEQ ID NO: 14 Of the amino acid sequence shown, the amino acid sequence corresponding to one or more specific regions is the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 6, the amino acid represented by SEQ ID NO: 10. Amino acid sequence which is replaced with amino acid sequence corresponding to one or more specific regions of the same kind contained in the sequence or amino acid sequence represented by SEQ ID NO: 18, or (v) amino acid sequence represented by SEQ ID NO: 18 Among them, the amino acid sequence corresponding to one or more specific regions is the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 0 In the amino acid sequence or SEQ ID NO represented: the same type of one or more amino acid sequences are replaced with the corresponding amino acid sequence in a specific region included in the amino acid sequence represented by 14; [b ^1] or less (i) To (v), the specific region is a complementarity determining region 1, a complementarity determining region 2, a complementarity determining region 3, a framework region 1, a framework region 2, a framework region 3, A protein characterized by being selected from the group consisting of framework regions 4, (i) of the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence corresponding to one or more specific regions is a sequence The amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 16, or the amino acid represented by SEQ ID NO: 20. An amino acid sequence exchanged with an amino acid sequence corresponding to one or more specific regions of the same kind contained in the sequence, (ii) an amino acid corresponding to one or more specific regions in the amino acid sequence represented by SEQ ID NO: 8 The same sequence contained in the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 16 or the amino acid sequence represented by SEQ ID NO: 20 Among the amino acid sequences represented by (iii) SEQ ID NO: 12, which are replaced with the amino acid sequences corresponding to one or more specific regions of the kind, the amino acid sequences corresponding to one or more specific regions are SEQ ID NOs. : 4, the amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence represented by SEQ ID NO: 16, or the amino acid sequence represented by SEQ ID NO: 20. Corresponding to one or more specific regions of the amino acid sequence represented by (iv) SEQ ID NO: 16 which is replaced with an amino acid sequence corresponding to one or more specific regions of the same type contained in the amino acid sequence. The amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO:
Amino acid sequence represented by 12 or the amino acid sequence represented by SEQ ID NO: 20, which is replaced with an amino acid sequence corresponding to one or more specific regions of the same type, or (v) SEQ ID NO: 20 In the amino acid sequence represented by, the amino acid sequence corresponding to one or more specific regions is the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO:
The amino acid sequence represented by 8 or the amino acid sequence represented by SEQ ID NO: 12 or the amino acid sequence represented by SEQ ID NO: 16 is replaced with an amino acid sequence corresponding to one or more specific regions of the same type. Amino acid sequence; [a ² ] amino acid sequence represented by SEQ ID NO: 2, amino acid sequence represented by SEQ ID NO: 6, amino acid sequence represented by SEQ ID NO: 10, amino acid sequence represented by SEQ ID NO: 14 , An amino acid sequence represented by SEQ ID NO: 18 or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the protein of [a ¹ ] and SEQ ID NO: 4 The amino acid sequence represented by, the amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 16, A protein that binds to female hormones when forming a complex with a protein having the amino acid sequence represented by SEQ ID NO: 20 or the amino acid sequence of the protein of [b ¹ ]; [b ² ] SEQ ID NO: 4 The amino acid sequence represented by, the amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 16, the amino acid sequence represented by SEQ ID NO: 20. Alternatively, it has an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the protein of [b ¹ ] and has the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 6 The amino acid sequence represented by, the amino acid sequence represented by SEQ ID NO: 10, the amino acid sequence represented by SEQ ID NO: 14, the amino acid sequence represented by SEQ ID NO: 18, or A protein that binds to female hormones when forming a complex with a protein having the amino acid sequence of the protein of [a ¹ ]; [a ³ ] the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 6 The amino acid sequence represented by SEQ ID NO: 10, the amino acid sequence represented by SEQ ID NO: 14, the amino acid sequence represented by SEQ ID NO: 18, or the amino acid sequence of the protein of [a ¹ ] An amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 8, or an amino acid sequence represented by SEQ ID NO: 12, which has an amino acid sequence in which one or more amino acids are deleted, substituted or added The amino acid sequence represented by SEQ ID NO: 16, the amino acid sequence represented by SEQ ID NO: 20, or the amino acid sequence of the protein of [b ¹ ] Or a protein which binds to female hormones when forming a complex with a protein having an amino acid sequence in which two or more amino acids are deleted, substituted or added; [b ³ ] represented by SEQ ID NO: 4 Amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 12, amino acid sequence represented by SEQ ID NO: 16, amino acid sequence represented by SEQ ID NO: 20, or b ¹ ] having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of the protein [ ¹ ], and the amino acid sequence represented by SEQ ID NO: 2 is represented by SEQ ID NO: 6. Amino acid sequence represented by SEQ ID NO: 10, amino acid sequence represented by SEQ ID NO: 14, amino acid sequence represented by SEQ ID NO: 18, or the amino acid sequence of [a ¹ ] A protein that binds to female hormones when forming a complex with a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the protein. 3. The protein or a salt thereof according to claim 1 or 2, wherein the female hormone is estrogen or a synthetic female hormone. 4. The protein or a salt thereof according to claim 3, wherein the estrogen is estradiol, estrone or estriol. 5. The protein or a salt thereof according to claim 3, wherein the synthetic female hormone is ethinyl estradiol or diethyl bestrol. 6. A method for gene recombination of the protein according to any one of claims 1 to 5. 7. A protein or a salt thereof obtained by the method according to claim 6. 8. A partial peptide of the protein according to any one of claims 1 to 5 or 7, or a salt thereof. 9. A polynucleotide encoding the protein or the partial peptide thereof according to any one of claims 1 to 5 and 7. 10. A recombinant vector containing the polynucleotide according to claim 9. 11. A transformant transformed with the recombinant vector according to claim 10. 12. A protein according to any one of claims 1 to 5 and 7, or a partial peptide thereof or a salt thereof is produced and collected, which is characterized in that it is collected.
5. A method for producing the protein according to any one of 5 to 7 or a partial peptide thereof or a salt thereof. 13. A complex formed by linking the protein of the following [a] and the protein of the following [b]: [a] the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 6, The amino acid sequence represented by SEQ ID NO: 10, the amino acid sequence represented by SEQ ID NO: 14, the amino acid sequence represented by SEQ ID NO: 18,
Or a protein having an amino acid sequence substantially the same as these; [b] the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence represented by SEQ ID NO: 12, the SEQ ID NO: A protein having the amino acid sequence represented by 16, the amino acid sequence represented by SEQ ID NO: 20, or an amino acid sequence substantially the same as these. 14. The complex according to claim 13, which is a single-chain antibody. 15. A phage which expresses the single chain antibody according to claim 14. 16. A method for identifying female hormones that bind to the complex, which comprises using the complex according to claim 13 or 14 or the phage according to claim 15. 17. A method for measuring or quantifying female hormones, which comprises using the complex according to claim 13 or 14, or the phage according to claim 15. 18. A kit for measuring or quantifying female hormones, which comprises the complex according to claim 13 or 14, or the phage according to claim 15. 19. A method for concentrating female hormones, which comprises using the complex according to claim 13 or 14, or the phage according to claim 15. 20. A kit for concentrating female hormones, which comprises the complex according to claim 13 or 14, or the phage according to claim 15.