JP6081699B2 - Method for analyzing IL-28B - Google Patents

Description

  The present invention relates to an anti-IL-28B monoclonal antibody and a method for specific analysis of IL-28B using the monoclonal antibody. According to the present invention, it is possible to analyze major IL-28B and minor IL-28B separately from IL-28A with high sensitivity. In addition, major IL-28B can be separated from minor IL-28B and measured with high sensitivity.

Hepatitis C virus (hereinafter sometimes referred to as HCV) is a blood-borne virus, and when HCV infection occurs, 20 to 30% is cured in an acute course, and 70 to 80% is sustained. Transition to infection. Many persistent infections caused by HCV become chronic hepatitis, and if not treated, most of them progress to cirrhosis and liver cancer.
As a treatment for this chronic hepatitis C, treatment with interferon-α (hereinafter sometimes referred to as IFN-α) was started in 1992. Thereafter, the combined administration of IFN-α and ribavirin was approved, and from 2004, the combined administration of PEGylated IFN-α and ribavirin was applied to insurance for intractable chronic hepatitis C with genotype 1b and high viral load. The marked efficiency of treatment has greatly improved.

Recently, regarding this combined administration of PEGylated IFN-α and ribavirin, Tanaka et al. Have a plurality of SNPs in the vicinity of IL-28B, and the therapeutic effect of combined administration of PEGylated IFN-α and ribavirin depends on the difference in alleles. Are reported to be different (Non-Patent Document 1).
IL-28B is encoded by a gene of about 1.5 Kb present on chromosome 19 and is also called IFN-λ3. There are cytokines having three similar structures of IFN-λ1, IFN-λ2, and IFN-λ3 in IFN-λ, which are also referred to as IL-29, IL-28A, and IL-28B, respectively (non-patented) Reference 4). These three cytokines IL-29, IL-28A, and IL-28B are known to exert an antiviral effect via a receptor different from IFN-α. For example, IL-29 is HCV Clinical trials have been started in Europe, and it is becoming clear that there are few side effects.

  The amino acid sequences of these IL-29, IL-28A and IL-28B proteins are highly homologous, the homology between IL-29 and IL-28A, and the homology between IL-29 and IL-28B is 81. Furthermore, the homology between IL-28A and IL-28B reaches 96% (Non-patent Document 4). Non-Patent Document 2 measures the concentrations of IL-29 and IL-28A in the serum of type C-infected patients by ELISA, and the measurement of IL-28A is based on the commercially available ELISA kit (Human IL-28A) of R & D Systems. / IFN-lamda 2 DuoSet). It is disclosed that the homology of the amino acid sequence between IL-28A and IL-28B is 96%, and the capture antibody used in the IL-28A ELISA kit has a significant cross-reactivity with IL-28B. (Non-Patent Document 2). Therefore, conventional IL-28A and IL-28B could not be measured separately.

“Nature Genetics” (UK) 2009, Vol. 41, p. 1105-1109 “Journal of Hepatology” (Switzerland) 2011, Vol. 54, p. 859-865 “PLoS One” (USA) 2010, Volume 5, e15200 “Ayumi of Medicine” (Japan) 2010, 234, p. 487-492

Furthermore, there is one SNP in the gene encoding IL-28B, and there is a major IL-28B translated from a major allele and a minor IL-28B translated from a minor allele. Specifically, in minor IL-28B, the 74th amino acid of major IL-28B is substituted from lysine (K) to arginine (R) (hereinafter, this substitution may be referred to as “K74R”).
Recently, an anti-IL-28B monoclonal antibody was obtained using this minor IL-28B as an immunogen, and an IL-28B specific ELISA kit using the obtained monoclonal antibody was commercially available from R & D.
However, the measurement sensitivity of this ELISA kit is a detection sensitivity of 100 pg / mL, as described in Comparative Examples described later, and the sensitivity is insufficient to measure IL-28B in human blood. It was considered. Moreover, there was no ELISA kit capable of specifically measuring major IL-28B and ELISA kit capable of specifically measuring minor IL-28B.
Accordingly, an object of the present invention is to provide an IL-28B specific analysis method capable of specifically and highly sensitively measuring IL-28B (IFN-λ3). Another object of the present invention is to provide a major IL-28B specific analysis method capable of specifically measuring major IL-28B.

As a result of intensive studies on an analytical method capable of measuring IL-28B specifically and with high sensitivity, the present inventors have found that a monoclonal antibody that recognizes a specific discontinuous epitope of IL-28B is a major IL- It was found to bind specifically to 28B and minor IL-28B. The major and minor IL-28B analysis method using this monoclonal antibody was found to be able to specifically measure major IL-28B and minor IL-28B.
Furthermore, it was found that a monoclonal antibody that recognizes another specific discontinuous epitope of IL-28B specifically binds to major IL-28B. Then, it was found that major IL-28B can be specifically measured by a major IL-28B analysis method using this monoclonal antibody.
The present invention is based on these findings.
Therefore, the present invention
[1] IL that binds to major IL-28B composed of the amino acid sequence represented by SEQ ID NO: 1 and minor IL-28B composed of the amino acid sequence represented by SEQ ID NO: 3 and comprises the amino acid sequence represented by SEQ ID NO: 5 Anti-IL-28B monoclonal antibody (hereinafter referred to as “anti-IL-28B monoclonal antibody (A)”) that does not bind to −28A and does not bind to the peptide fragment consisting of the amino acid sequence represented by SEQ ID NOs: 8-26. Or an antigen-binding fragment thereof,
[2] (1) Polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 28, heavy chain complementarity determining consisting of amino acids 50 to 66 A polypeptide of region 2, a heavy chain variable region domain comprising a polypeptide of heavy chain complementarity determining region 3 consisting of amino acids 99 to 113, and amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 30 Polypeptide of light chain complementarity determining region 1 consisting of amino acids, polypeptide of light chain complementarity determining region 2 consisting of amino acids 54 to 60, light chain complementarity determining region 3 consisting of amino acids 93 to 101 A light chain variable region domain comprising the polypeptide of: or (2) a heavy chain complementarity determining region consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 32 A heavy chain variable region comprising a polypeptide of 1 heavy chain complementarity determining region 2 consisting of amino acids 50 to 66, a polypeptide of heavy chain complementarity determining region 3 consisting of amino acids 99 to 109 A polypeptide of light chain complementarity determining region 1 consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 34, and light chain complementarity determining region 2 consisting of amino acids 54 to 60 A monoclonal antibody according to [1], which has a light chain variable region domain comprising a polypeptide, a light chain complementarity determining region 3 polypeptide consisting of amino acids 93 to 101, or an antigen-binding fragment thereof,
[3] The monoclonal antibody according to [1], which binds to an epitope to which the monoclonal antibody according to [2] binds, or an antigen-binding fragment thereof,
[4] A major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1 and an IL consisting of the minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3 and the amino acid sequence represented by SEQ ID NO: 5 Anti-IL-28B monoclonal antibody (hereinafter referred to as “anti-IL-28B monoclonal antibody (B)”) that does not bind to -28A and does not bind to a peptide fragment consisting of the amino acid sequence represented by SEQ ID NOs: 8-26. Or an antigen-binding fragment thereof,
[5] (1) Heavy chain complementarity determining region 1 polypeptide consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 36, heavy chain complementarity determining consisting of amino acids 50 to 66 A polypeptide of region 2, a heavy chain variable region domain comprising a polypeptide of heavy chain complementarity determining region 3 consisting of amino acids 99 to 106, and amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 38 Polypeptide of light chain complementarity determining region 1 consisting of amino acids, polypeptide of light chain complementarity determining region 2 consisting of amino acids 54 to 60, light chain complementarity determining region 3 consisting of amino acids 93 to 101 A light chain variable region domain comprising the polypeptide of;
(2) Polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 40, heavy chain complementarity determining region 2 consisting of amino acids 50 to 66 A polypeptide, a heavy chain variable region domain comprising the heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 106, and amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 42 Polypeptide of light chain complementarity determining region 1, polypeptide of light chain complementarity determining region 2 consisting of amino acids 54 to 60, polypeptide of light chain complementarity determining region 3 consisting of amino acids 93 to 101 Or (3) a heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 44 A heavy chain variable region domain comprising a heavy chain complementarity determining region 2 polypeptide consisting of amino acids 50 to 66, a heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 106, and A polypeptide of the light chain complementarity determining region 1 consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 46; a polypeptide of the light chain complementarity determining region 2 consisting of amino acids 54 to 60; A monoclonal antibody according to [4], which has a light chain variable region domain comprising a polypeptide of light chain complementarity determining region 3 consisting of amino acids 93 to 101, or an antigen-binding fragment thereof,
[6] The monoclonal antibody according to [4], which binds to an epitope to which the monoclonal antibody according to [5] binds, or an antigen-binding fragment thereof,
[7] A hybridoma that produces the monoclonal antibody according to any one of [1] to [6],
[8] A step of bringing the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [3] into contact with a test sample that may contain IL-28B. , Major and minor IL-28B analysis method (hereinafter sometimes referred to as “IL-28B analysis method (A)”),
[9] (1) contacting the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [3] with a test sample that may contain IL-28B; and (2) Major and minor IL-28B analysis method according to [8], comprising the step of contacting a test sample with a second antibody that binds to IL-28B;
[10] Major IL-28B comprising the amino acid sequence represented by SEQ ID NO: 1, minor IL-28B comprising the amino acid sequence represented by SEQ ID NO: 3, and SEQ ID NO: The major and minor IL-28B according to [8] or [9], which is an anti-IL-28A / B monoclonal antibody, or an antigen-binding fragment thereof, that binds to IL-28A consisting of the amino acid sequence represented by 5 Analysis method,
[11] The anti-IL-28A / B monoclonal antibody is a polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 48; Polypeptide of heavy chain complementarity determining region 2 consisting of amino acids, heavy chain variable region domain comprising heavy chain complementarity determining region 3 consisting of amino acids 99 to 110, and 24 in the amino acid sequence represented by SEQ ID NO: 50 Polypeptide of light chain complementarity determining region 1 consisting of amino acids Nos. To 38, polypeptide of light chain complementarity determining region 2 consisting of amino acids 54 to 60, and light chain consisting of amino acids 93 to 101 The major and minor I according to [10], which is a monoclonal antibody having a light chain variable region domain comprising a polypeptide of complementarity determining region 3 -28B analysis method,
[12] A step of bringing the monoclonal antibody or antigen-binding fragment thereof according to any one of [4] to [6] into contact with a test sample that may contain IL-28B. , Major IL-28B analysis method (hereinafter sometimes referred to as "IL-28B analysis method (B)"),
[13] (1) contacting the monoclonal antibody or antigen-binding fragment thereof according to any one of [4] to [6] with a test sample that may contain IL-28B; and (2) Contacting a test sample with a second antibody that binds to IL-28B;
A major IL-28B analysis method according to [12],
[14] The second antibody that binds to IL-28B is (1) the monoclonal antibody or the antigen-binding fragment thereof according to any one of [1] to [3], or (2) SEQ ID NO: 1 Anti-IL that binds to major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3, minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3, and IL-28A consisting of the amino acid sequence represented by SEQ ID NO: 5. A major IL-28B analysis method according to [12] to [13], which is a -28A / B monoclonal antibody, or an antigen-binding fragment thereof,
[15] The anti-IL-28A / B monoclonal antibody is a polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 48; Polypeptide of heavy chain complementarity determining region 2 consisting of amino acids, heavy chain variable region domain comprising heavy chain complementarity determining region 3 consisting of amino acids 99 to 110, and 24 in the amino acid sequence represented by SEQ ID NO: 50 Polypeptide of light chain complementarity determining region 1 consisting of amino acids Nos. To 38, polypeptide of light chain complementarity determining region 2 consisting of amino acids 54 to 60, and light chain consisting of amino acids 93 to 101 Major IL-28B analysis according to [14], which is a monoclonal antibody having a light chain variable region domain comprising a polypeptide of complementarity determining region 3 Law,
[16] (a) The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [3], and (b) Major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: An anti-IL-28A / B monoclonal antibody that binds to a minor IL-28B consisting of the amino acid sequence represented by 3 and IL-28A consisting of the amino acid sequence represented by SEQ ID NO: 5, or an antigen-binding fragment thereof. Including major and minor IL-28B analysis kits (hereinafter sometimes referred to as "IL-28B analysis kit (A)"),
[17] The anti-IL-28A / B monoclonal antibody is a polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 48; Polypeptide of heavy chain complementarity determining region 2 consisting of amino acids, heavy chain variable region domain comprising heavy chain complementarity determining region 3 consisting of amino acids 99 to 110, and 24 in the amino acid sequence represented by SEQ ID NO: 50 Polypeptide of light chain complementarity determining region 1 consisting of amino acids Nos. To 38, polypeptide of light chain complementarity determining region 2 consisting of amino acids 54 to 60, and light chain consisting of amino acids 93 to 101 The major and minor IL according to [16], which is a monoclonal antibody having a light chain variable region domain comprising a polypeptide of complementarity determining region 3 28B analysis kit,
[18] The monoclonal antibody according to any one of (a) [4] to [6], or an antigen-binding fragment thereof, and the monoclonal antibody according to any one of (b) (1) [1] to [3] An antibody or antigen-binding fragment thereof, or (2) a major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1, a minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3, and a sequence represented by SEQ ID NO: 5. A major IL-28B assay kit (hereinafter referred to as “IL-28B assay kit (B)”, which comprises an anti-IL-28A / B monoclonal antibody or an antigen-binding fragment thereof that binds to IL-28A comprising the amino acid sequence of ) "),
[19] The anti-IL-28A / B monoclonal antibody is a polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 48; Polypeptide of heavy chain complementarity determining region 2 consisting of amino acids, heavy chain variable region domain comprising heavy chain complementarity determining region 3 consisting of amino acids 99 to 110, and 24 in the amino acid sequence represented by SEQ ID NO: 50 Polypeptide of light chain complementarity determining region 1 consisting of amino acids Nos. To 38, polypeptide of light chain complementarity determining region 2 consisting of amino acids 54 to 60, and light chain consisting of amino acids 93 to 101 Major IL-28B analysis according to [18], which is a monoclonal antibody having a light chain variable region domain comprising a polypeptide of complementarity determining region 3 Kit,
About.

According to the anti-IL-28B monoclonal antibody (A) of the present invention, since it binds to major IL-28B and minor IL-28B without binding to IL-28A, major and minor IL-28B are specifically detected. can do. Furthermore, the IL-28B analysis method (A) of the present invention can specifically analyze major and minor IL-28B by using the monoclonal antibody. In addition, the IL-28B analysis method (A) of the present invention can measure major and minor IL-28B to 0.1 pg / mL with high sensitivity.
In addition, according to the anti-IL-28B monoclonal antibody (B) of the present invention, since it binds to major IL-28B without binding to IL-28A and minor IL-28B, major IL-28B is specifically detected. can do. Furthermore, the IL-28B analysis method (B) of the present invention can specifically analyze major IL-28B by using the monoclonal antibody (B). Further, the IL-28B analysis method (B) of the present invention can measure major IL-28B up to 0.1 pg / mL with high sensitivity.

It is the figure which compared the amino acid sequence of major IL-28B, minor IL-28B (K74R), and IL-28A protein. TA2602 monoclonal antibody (A), TA2664 monoclonal antibody (B), TA2613 monoclonal antibody (C), and TA2650 monoclonal antibody (D) rIL-28B Wild (major IL-28B), rIL-28B K74R (minor IL-28B) It is a graph which shows the result of ELISA which investigated the coupling | bonding of rIL-28A. By major and minor IL-28B analysis methods using TA2650 as a solid phase antibody and TA2664 as a labeled antibody, rIL-28B W (HeLa) (major IL-28B), rIL-28B W (Invitrogen) (major IL-28B ), RIL-28B K74R (HeLa) (minor IL-28B), rIL-28B K74R (R & D) (minor IL-28B), rIL-28A (R & D), and rIL-29 (R & D) are shown. It is a graph. By major and minor IL-28B analysis methods using TA2664 as a solid phase antibody and TA2650 as a labeled antibody, rIL-28BW (HeLa) (major IL-28B), rIL-28BW (Invitrogen) (major IL-28B ), RIL-28B K74R (HeLa) (minor IL-28B), rIL-28B K74R (R & D) (minor IL-28B), rIL-28A (R & D), and rIL-29 (R & D) are shown. It is a graph. According to a major IL-28B analysis method using TA2602 as a solid phase antibody and TA2664 as a labeled antibody, rIL-28BW (HeLa) (major IL-28B), rIL-28BW (Invitrogen) (major IL-28B), The graph which shows the result of having measured rIL-28B K74R (HeLa) (minor IL-28B), rIL-28B K74R (R & D) (minor IL-28B), rIL-28A (R & D), and rIL-29 (R & D) is there. By a major IL-28B analysis method using TA2602 as a solid phase antibody and TA2650 as a labeled antibody, rIL-28BW (HeLa) (major IL-28B), rIL-28BW (Invitrogen) (major IL-28B), The graph which shows the result of having measured rIL-28B K74R (HeLa) (minor IL-28B), rIL-28B K74R (R & D) (minor IL-28B), rIL-28A (R & D), and rIL-29 (R & D) is there. Using IL-28B specific ELISA kit of R & D, rIL-28B W (Invitrogen) (major IL-28B), rIL-28B K74R (R & D) (minor IL-28B), rIL-28B attached to the kit It is a graph which shows the result of having measured (DuoSet) (minor IL-28B), rIL-28A (R & D), and rIL-29 (R & D).

[1] Anti-IL-28B monoclonal antibody The anti-IL-28B monoclonal antibody of the present invention is an anti-IL-28B monoclonal antibody that binds to IL-28B but does not bind to IL-28A.
Specifically, the anti-IL-28B monoclonal antibody binds to (A) major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1 and minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3. An anti-IL-28B monoclonal antibody that does not bind to IL-28A consisting of the amino acid sequence represented by SEQ ID NO: 5 and does not bind to the peptide fragment consisting of the amino acid sequence represented by SEQ ID NO: 8 to 26 (anti-IL- 28B monoclonal antibody (A)) and (B) major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1 and minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3 and SEQ ID NO: 5 It does not bind to IL-28A consisting of the amino acid sequence represented by and consists of the amino acid sequence represented by SEQ ID NOs: 8-26 It does not bind to peptide fragments, including anti-IL-28B monoclonal antibody (anti-IL-28B monoclonal antibody (B)).

(IL-28B)
IL-28B is a protein consisting of 200 amino acids encoded by an approximately 1.5 Kb gene present on chromosome 19 and is also referred to as IFN-λ3. IL-28B has 25 signal peptides at the N-terminus, and IL-28B secreted outside the cell is a peptide consisting of 26 to 200 amino acids by cleavage of the signal peptide. The monoclonal antibody of the present invention is a monoclonal antibody that binds to a peptide consisting substantially of 26-200 amino acids of IL-28B.
In addition to IFN-λ3, the chromosome 19 also includes IFN-λ1 (IL-29) and IFN-λ2 (IL-28A), and IFN-λ1, IFN-λ2, and IFN-λ3 are collectively It is called the IFN-λ family.
The gene encoding IL-28B has a SNP at position 221 of the translation region, the major allele is A, and the minor allele is G. The 74th amino acid of major IL-28B translated from the major allele is lysine (K), and the 74th amino acid of minor IL-28B translated from the minor allele is arginine (R). In the present specification, substitution of lysine (K) from major IL-28B to arginine (R) from minor IL-28B may be referred to as “K74R” substitution.
In the amino acid sequence of minor IL-28B, the 10th threonine (T) is replaced with methionine (M) (hereinafter referred to as “T10M” substitution), and the 32nd histidine ( H) is substituted with arginine (R) (hereinafter referred to as “H32R” substitution), 96th methionine (M) is substituted with valine (V) (hereinafter referred to as “M96V” substitution), and 120th Valine (V) is substituted with glycine (G) (hereinafter referred to as “V120G” substitution), 137th phenylalanine (F) is substituted with leucine (L) (hereinafter referred to as “F137L” substitution), 160 The second tyrosine (Y) is substituted with histidine (H) (hereinafter referred to as “Y160H” substitution) (FIG. 1). Therefore, the amino acid sequence homology between minor IL-28B and IL-28A is 97%.
In the amino acid sequence of major IL-28B, the 74th arginine (R) is substituted with lysine (K) in addition to the above-mentioned substitution of minor IL-28B with respect to the amino acid sequence of IL-28A (hereinafter referred to as “R74K”). (Referred to as “substitution”) (FIG. 1). Therefore, the amino acid sequence homology between major IL-28B and IL-28A is 96.5%.
The amino acid sequence homology between major IL-28B and minor IL-28B is 99.5%.

  That is, the difference in amino acid sequence between minor IL-28B and IL-28A is only 6 amino acids, and the difference in amino acid sequence between major IL-28B and IL-28A is only 7 amino acids. The difference in amino acid sequence from major IL-28B is only 1 amino acid.

[1-1] Anti-IL-28B monoclonal antibody (A)
The anti-IL-28B monoclonal antibody (A) of the present invention binds to major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1 and minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3, It is an anti-IL-28B monoclonal antibody that does not bind to IL-28A consisting of the amino acid sequence represented by No. 5 and does not bind to the peptide fragment consisting of the amino acid sequence represented by SEQ ID Nos. 8-26. An antigen-binding fragment of the anti-IL-28B monoclonal antibody (A) can also be used in the same manner as the antibody.

  As used herein, “does not bind to IL-28A” means that when used as an antibody, binding to IL-28A is at a level that does not substantially affect the binding. More specifically, the binding to IL-28A is 1/100 or less, preferably 1/300 or less, more preferably 1/500 or less, and most preferably 1/1000 or less with respect to the binding of major IL-28B. It means that there is. In addition, “the binding to IL-28A is 1/100 or less with respect to the binding of major IL-28B” means that, for example, major IL-28B or IL-28A was immobilized as shown in the Examples. In ELISA, it means that the concentration of monoclonal antibody that can obtain the same OD value is 100 times higher than that of IL-28A.

  The anti-IL-28B monoclonal antibody (A) of the present invention is a group of monoclonal antibodies that can bind to a specific epitope, and typical antibodies include TA2613 and TA2664. The monoclonal antibodies contained in the anti-IL-28B monoclonal antibody (A) have the same or highly homologous CDRs as described later, recognize the same epitope, and have similar properties to each other. . In addition, the monoclonal antibody belonging to the anti-IL-28B monoclonal antibody (A) can be obtained according to the description in Examples described later.

(Epitope A)
The epitope (hereinafter referred to as epitope (A)) to which the anti-IL-28B monoclonal antibody (A) of the present invention binds is an epitope that is present in major IL-28B and minor IL-28B and not present in IL-28A. In addition, it is an epitope that does not exist in a 20-amino acid synthetic polypeptide (SEQ ID NO: 8-26) that overlaps by 10 amino acids synthesized based on the amino acid sequence of major IL-28B (SEQ ID NO: 1). That is, the epitope (A) is a discontinuous epitope (conformational epitope) that exists in major IL-28B and minor IL-28B and does not exist in IL-28A.
The epitope (A) is an epitope that is not present in IL-28A, but from the “T10M” substitution, “H32R” substitution, “M96V” substitution, “V120G” substitution, “F137L” substitution, and “Y160H” substitution. It is a discontinuous epitope (conformational epitope) formed in major IL-28B and minor IL-28B by one selected substitution or a combination of two or more substitutions.
More specifically, it is a discontinuous epitope (conformational epitope) to which a TA2613 monoclonal antibody or TA2664 having a specific CDR can bind.

  The anti-IL-28B monoclonal antibody (A) is obtained by using a recombinant protein having an amino acid sequence of amino acids 26 to 200 of IL-28B (hereinafter sometimes referred to as rIL-28B (26-200)) as an immunogen. TA2613 and TA2664. TA2613 and TA2664 were almost completely inhibited from binding to rIL-28B (26-200) by the epitope competition test of the Examples. Therefore, monoclonal antibodies TA2613 and TA2664 are monoclonal antibodies that recognize the same discontinuous epitope.

(Variable region domain)
The anti-IL-28B monoclonal antibody (A) of the present invention binds to the epitope A. The anti-IL-28B monoclonal antibody (A) that binds to the epitope A has the same or highly homologous CDR. Yes.
From the amino terminus, the heavy chain consists of a variable region polypeptide (hereinafter referred to as heavy chain variable region domain (VH)) and a constant domain three domain polypeptide, namely heavy chain constant region domain 1 (CH1), heavy chain constant. It has normal region domain 2 (CH2) and heavy chain constant region domain 3 (CH3) in that order. The heavy chain variable region domain has three complementarity determining regions, namely, a heavy chain complementarity determining region 1 (hereinafter sometimes referred to as H-CDR1) and a heavy chain complementarity determining region 2 (hereinafter referred to as H- And a heavy chain complementarity determining region 3 (hereinafter also referred to as H-CDR3), and these three complementarity determining regions are surrounded by a heavy chain variable region framework. Yes. Specifically, the heavy chain variable region framework consists of four framework region polypeptides, namely, H-FR1, H-FR2, H-FR3, and H-FR4 from the amino terminus. Thus, the heavy chain variable region domain includes H-FR1, H-CDR1, H-FR2, H-CDR2, H-FR3, H-CDR3, and H-FR4 in that order.

On the other hand, the light chain comprises a variable region polypeptide (hereinafter referred to as light chain variable region domain (VL)) and a constant region polypeptide (hereinafter referred to as light chain constant region domain (CL)) from the amino terminus. Have in order. The light chain variable region domain has three complementarity determining regions, namely, a light chain complementarity determining region 1 (hereinafter sometimes referred to as L-CDR1) and a light chain complementarity determining region 2 (hereinafter referred to as L- And light chain complementarity determining region 3 (hereinafter sometimes referred to as L-CDR3), and these three complementarity determining regions are surrounded by a light chain variable region framework. Yes. The light chain variable region framework is specifically composed of four framework region polypeptides, namely, L-FR1, L-FR2, L-FR3, and L-FR4 from the amino terminus. Thus, the light chain variable region domain contains the respective polypeptides of L-FR1, L-CDR1, L-FR2, L-CDR2, L-FR3, L-CDR3, and L-FR4 in that order.
Polypeptides consisting of amino acid sequences constituting each domain in the heavy chain and light chain variable region polypeptides are assigned by Kabat (1991), and / or Chothia and Lesk, J. Mol. Biol. 196: 901. -917 (1987); Chothia et al., Nature 342: 878-883 (1989).

(CDR of TA2613)
The heavy chain variable region domain of TA2613 of the present invention is preferably a polypeptide (SYGMS) of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 28, 50 to A heavy chain variable region domain comprising a heavy chain complementarity determining region 2 polypeptide consisting of amino acid 66 (TITGGGSYTYYPDGVKG) and a heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 113 (RGDYGSNYLYWYFDV). And most preferably a heavy chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 28. The light chain variable region domain is preferably a light chain complementarity determining region 1 polypeptide (RASKSVSTSGYSYMH) consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 30, 54 to 60 A light chain variable region domain comprising a polypeptide of light chain complementarity determining region 2 consisting of amino acids of (LVSNLES) and a polypeptide of light chain complementarity determining region 3 consisting of amino acids 93 to 101 (QHIRELTRS), Most preferred is a light chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 30.

(CDR of TA2664)
The heavy chain variable region domain of TA2664 of the present invention is preferably a polypeptide (DYYMY) of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 32, 50 to A heavy chain variable region domain comprising a heavy chain complementarity determining region 2 polypeptide consisting of amino acid 66 (YISNGGGSTNYPDTVKG) and a heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 109 (PSLLRSAWFAY). And most preferably a heavy chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 32. The light chain variable region domain is preferably a light chain complementarity determining region 1 polypeptide (RASKSVSTSGYSYMH) consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 34, 54 to 60 A light chain variable region domain comprising a polypeptide of light chain complementarity determining region 2 consisting of amino acids of (LVSNLES) and a polypeptide of light chain complementarity determining region 3 consisting of amino acids 93 to 101 (QHIRELTRS), Most preferred is a light chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 34.

(Monoclonal antibody binding to epitope (A))
The anti-IL-28B monoclonal antibody (A) of the present invention includes a monoclonal antibody that binds to the epitope (A). The monoclonal antibody that binds to epitope (A) binds to major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1 and minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3, An anti-IL-28B monoclonal antibody that does not bind to IL-28A consisting of the amino acid sequence shown and does not bind to a peptide fragment consisting of the amino acid sequence shown in SEQ ID NOs: 8-26, and is the same as TA2613 or TA2664 It has properties. In order to bind to the epitope (A), it preferably has a CDR identical to or highly homologous to TA2613 or TA2664.
Whether or not it can bind to the epitope (A) can be determined by an epitope competition test described in the Examples using an antibody having CDR of TA2613 or TA2664.
The monoclonal antibody that binds to the epitope (A) of the present invention can be used in the major and minor IL-28B specific analysis method (A).

[1-2] Anti-IL-28B monoclonal antibody (B)
The anti-IL-28B monoclonal antibody (B) of the present invention binds to major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1 and minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3 and the sequence It is an anti-IL-28B monoclonal antibody that does not bind to IL-28A consisting of the amino acid sequence represented by No. 5 and does not bind to the peptide fragment consisting of the amino acid sequence represented by SEQ ID Nos. 8-26. An antigen-binding fragment of anti-IL-28B monoclonal antibody (B) can also be used in the same manner as the antibody.

  As used herein, “does not bind to minor IL-28B” means that when used as an antibody, binding to minor IL-28B is at a level that does not substantially affect the binding. More specifically, the binding to minor IL-28B relative to the binding of major IL-28B is 1/100 or less, preferably 1/300 or less, more preferably 1/500 or less, most preferably 1/1000 or less. It means that. In addition, “the binding to minor IL-28B is 1/100 or less relative to the binding of major IL-28B” means that, for example, major IL-28B or minor IL-28B is immobilized on a solid phase as shown in the Examples. This means that the concentration of the monoclonal antibody that can obtain the same OD value is 100 times higher in the minor IL-28B in the modified ELISA.

  The anti-IL-28B monoclonal antibody (B) of the present invention is a group of monoclonal antibodies capable of binding to a specific epitope, and representative examples include TA2601, TA2602, and TA2603. The monoclonal antibodies contained in the anti-IL-28B monoclonal antibody (B) have the same or highly homologous CDRs as described later, recognize the same epitope, and have similar properties to each other. . In addition, a monoclonal antibody belonging to the anti-IL-28B monoclonal antibody (B) can be obtained according to the description in Examples described later.

(Epitope B)
The epitope to which the anti-IL-28B monoclonal antibody (B) of the present invention binds (hereinafter referred to as epitope (B)) is an epitope that is present in major IL-28B and not present in IL-28A and minor IL28, and It is an epitope that does not exist in a 20-amino acid synthetic polypeptide (SEQ ID NO: 8 to 26) that overlaps by 10 amino acids synthesized based on the amino acid sequence of major IL-28B (SEQ ID NO: 1). That is, the epitope (B) is a discontinuous epitope (conformational epitope) that exists in major IL-28B and does not exist in IL-28A and minor IL-28B.
The epitope (B) is an epitope that is not present in IL-28A, but the “T10M” substitution, “H32R” substitution, “K74R” substitution, “M96V” substitution, “V120G” substitution, “F137L” substitution, and It is a discontinuous epitope (conformational epitope) formed in major IL-28B by one substitution selected from the “Y160H” substitution, or a combination of two or more substitutions.
More specifically, it is a discontinuous epitope (conformational epitope) to which a TA2601 monoclonal antibody, TA2602 monoclonal antibody, or TA2603 monoclonal antibody having a specific CDR can bind.

  Examples of the anti-IL-28B monoclonal antibody (B) include TA2601, TA2602, and TA2603 obtained using rIL-28B (26-200) containing no signal peptide as an immunogen. TA2601, TA2602, and TA2603 were almost completely inhibited from binding to rIL-28B (26-200) by the epitope competition test of the Examples. Therefore, monoclonal antibodies TA2601, TA2602, and TA2603 are monoclonal antibodies that recognize the same discontinuous epitope.

  Furthermore, the binding between the anti-IL-28B monoclonal antibody (A) and the anti-IL-28B monoclonal antibody (B) was partially inhibited in the epitope competition test of Examples. Therefore, it is considered that the epitope (A) and the epitope (B) exist at related positions.

(Variable region domain)
The anti-IL-28B monoclonal antibody (B) of the present invention binds to the epitope B. The anti-IL-28B monoclonal antibody (B) that binds to the epitope B has the same or highly homologous CDR. Yes.

(CDR of TA2601)
The heavy chain variable region domain of TA2601 of the present invention is preferably a polypeptide (DTYIH) of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 36, 50 to A heavy chain variable region domain comprising a heavy chain complementarity determining region 2 polypeptide consisting of amino acid 66 (RIDPANGNTKYDPKFQG) and a heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 106 (YGYDYFDY). And most preferably a heavy chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 36. The light chain variable region domain is preferably a light chain complementarity determining region 1 polypeptide (RASKSVSTSGYSYMH) consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 38, 54 to 60 A light chain variable region domain comprising a polypeptide of light chain complementarity determining region 2 consisting of amino acids of (LVSNLES) and a polypeptide of light chain complementarity determining region 3 consisting of amino acids 93 to 101 (QHIRELTRS), Most preferred is a light chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 38.

(CDR of TA2602)
The heavy chain variable region domain of TA2602 of the present invention is preferably a heavy chain complementarity determining region 1 polypeptide (DTYIH) consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 40, 50 to A heavy chain variable region domain comprising a heavy chain complementarity determining region 2 polypeptide consisting of amino acid 66 (RIDPANGNTKYDPKFQG) and a heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 106 (YGYDYFDY). And most preferably a heavy chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 40. The light chain variable region domain is preferably a light chain complementarity determining region 1 polypeptide (RASKSVSTSGYSYMH) consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 42, 54 to 60 A light chain variable region domain comprising a polypeptide of light chain complementarity determining region 2 consisting of amino acids of (LVSNLES) and a polypeptide of light chain complementarity determining region 3 consisting of amino acids 93 to 101 (QHIRELTRS), Most preferred is a light chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 42.

(CDR of TA2603)
The heavy chain variable region domain of TA2603 of the present invention is preferably a polypeptide (DTYIH) of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 44, 50 to A heavy chain variable region domain comprising a heavy chain complementarity determining region 2 polypeptide consisting of amino acid 66 (RIDPANGNIKYDPKFQG) and a heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 106 (YGYDYFDY). And most preferably a heavy chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 44. The light chain variable region domain is preferably a light chain complementarity determining region 1 polypeptide (RASKSVSTSGYSYMH) consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 46, 54 to 60 A light chain variable region domain comprising a polypeptide of light chain complementarity determining region 2 consisting of amino acids of (LVSNLES) and a polypeptide of light chain complementarity determining region 3 consisting of amino acids 93 to 101 (QHIRELTRS), Most preferred is a light chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 46.

(Monoclonal antibody binding to epitope (B))
The anti-IL-28B monoclonal antibody (B) of the present invention includes a monoclonal antibody that binds to the epitope (B). The monoclonal antibody that binds to epitope (B) binds to major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1 and minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3 and SEQ ID NO: 5 An anti-IL-28B monoclonal antibody that does not bind to IL-28A consisting of the amino acid sequence shown below and does not bind to the peptide fragment consisting of the amino acid sequence represented by SEQ ID NOs: 8-26, and said TA2601, TA2602, or TA2603 Have the same properties. In order to bind to the epitope (B), it preferably has a CDR that is the same as or highly homologous to TA2601, TA2602, or TA2603.
Whether or not it can bind to the epitope ( B ) can be determined by an epitope competition test described in Examples using antibodies having CDRs of TA2601, TA2602, and TA2603.
The monoclonal antibody that binds to the epitope (B) of the present invention can be used in the major and minor IL-28B specific analysis method (B).

(Antigen-binding fragment)
The antigen-binding fragment of the present invention was formed from Fab, Fab ′, F (ab ′) ₂ , and Fv fragments of the anti-IL-28B monoclonal antibody, as well as diabodies, single chain antibody molecules, and antibody fragments. By multispecific antibody is meant. These antigen-binding fragments can be obtained, for example, by digesting an antibody with a proteolytic enzyme (for example, pepsin or papain) by a conventional method, and subsequently purifying the protein by a conventional method for separating and purifying proteins. Or can be prepared by genetic recombination.

(Affinity constant)
The affinity constant of the anti-IL-28B monoclonal antibody of the present invention is not particularly limited, but preferably has an affinity constant of at least 10 ⁵ to 10 ⁹ M ⁻¹ , and most preferably an affinity constant of 10 ⁶ or more. It is what has. Binding affinity can be measured, for example, by the Scatchard assay of Munson et al., Anal. Biochem. 107: 220 (1980).

[2] IL-28B Analysis Method The IL-28B analysis method of the present invention is an analysis method that specifically measures IL-28B and does not measure IL-28A.
Specifically, the IL-28B analysis method comprises the steps of (A) contacting the anti-IL-28B monoclonal antibody (A) or an antigen-binding fragment thereof with a test sample that may contain IL-28B; A major and minor IL-28B analysis method (IL-28B analysis method (A)), and (B) the anti-IL-28B monoclonal antibody (B) or an antigen-binding fragment thereof, A major IL-28B analysis method (IL-28B analysis method (B)), characterized by comprising contacting with a test sample that may contain -28B.

[2-1] IL-28B analysis method (A)
In the IL-28B analysis method (A) of the present invention, the anti-IL-28B monoclonal antibody (A) described in the section “[1-1] Anti-IL-28B monoclonal antibody (A)” is used.
In addition, the IL-28B analysis method (A) of the present invention is not limited, but preferably (1) the anti-IL-28B monoclonal antibody (A) or an antigen-binding fragment thereof is treated with IL-28B. Contacting with a test sample that may be included; and (2) contacting the test sample with a second antibody that binds to IL-28B.
In the IL-28B analysis method (A), the second antibody is not limited as long as it binds to IL-28B, but is preferably major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 An anti-IL-28A / B monoclonal antibody, or an antigen-binding fragment thereof, that binds to minor IL-28B consisting of the amino acid sequence represented by the above and IL-28A consisting of the amino acid sequence represented by SEQ ID NO: 5. The anti-IL-28A / B monoclonal antibody is more preferably an anti-IL-28A / B monoclonal antibody (hereinafter referred to as anti-IL-28A / B) that does not bind to a peptide fragment consisting of the amino acid sequence represented by SEQ ID NOs: 8-26. Monoclonal antibody (C)).

  The anti-IL-28A / B monoclonal antibody (C) is preferably a polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 48, 50 to 66 Polypeptide of heavy chain complementarity determining region 2 consisting of amino acid No., heavy chain variable region domain comprising heavy chain complementarity determining region 3 consisting of amino acids 99 to 110, and amino acid sequence represented by SEQ ID NO: 50 A light chain complementarity determining region 1 polypeptide consisting of amino acids 24 to 38, a light chain complementarity determining region 2 polypeptide consisting of amino acids 54 to 60, and amino acids 93 to 101 A monoclonal antibody having a light chain variable region domain comprising a polypeptide of light chain complementarity determining region 3.

[2-2] IL-28B analysis method (B)
In the IL-28B analysis method (B) of the present invention, the anti-IL-28B monoclonal antibody (B) described in the section “[1-2] Anti-IL-28B monoclonal antibody (B)” is used.
Moreover, the IL-28B analysis method (B) of the present invention is not limited, but preferably (1) the anti-IL-28B monoclonal antibody (B) or an antigen-binding fragment thereof is treated with IL-28B. And (2) contacting the test sample with a second antibody that binds to IL-28B.
In the IL-28B analysis method (B), the second antibody is not limited as long as it binds to IL-28B, but preferably the second antibody is (1) the anti-IL-28B monoclonal antibody (A) or (2) major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1, minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3, and SEQ ID NO: 5. An anti-IL-28A / B monoclonal antibody, or an antigen-binding fragment thereof, that binds to IL-28A having the amino acid sequence shown.
More preferably, the anti-IL-28A / B monoclonal antibody does not bind to a peptide fragment consisting of the amino acid sequence represented by SEQ ID NOs: 8-26, according to the above-mentioned “[2-1] IL-28B analysis method (A)”. Anti-IL-28A / B monoclonal antibody (C) described in the above item.

<< Common Embodiment for IL-28B Analysis Methods (A) and (B) >>
The IL-28B specific analysis method of the present invention can further include a step of detecting a conjugate of IL-28B and the monoclonal antibody or the like (hereinafter sometimes referred to as a detection step).
In this specification, “analysis” means both “detection” for determining the presence or absence and “quantification (measurement)” for determining the amount.

  The anti-IL-28A / B monoclonal antibody (C) is a group of monoclonal antibodies capable of binding to a specific epitope, and TA2650 can be mentioned as a representative antibody. Monoclonal antibodies contained in the anti-IL-28A / B monoclonal antibody (C) have the same or highly homologous CDRs as described below, recognize the same epitope, and have similar properties to each other It is. In addition, the monoclonal antibody belonging to the anti-IL-28A / B monoclonal antibody (C) can be obtained according to the description in Examples described later.

(Epitope C)
The epitope to which the anti-IL-28A / B monoclonal antibody (C) of the present invention binds (hereinafter referred to as epitope (C)) is present in major IL-28B, minor IL-28B, and IL-28A, and It is an epitope that does not exist in a 20-amino acid synthetic polypeptide (SEQ ID NO: 8 to 26) that overlaps by 10 amino acids synthesized based on the amino acid sequence of IL-28B (SEQ ID NO: 1). That is, epitope (C) is a discontinuous epitope (conformational epitope) present in major IL-28B, minor IL-28B, and IL-28A. More specifically, it is a discontinuous epitope (conformational epitope) to which a TA2650 monoclonal antibody having a specific CDR can bind.

Examples of the anti-IL-28A / B monoclonal antibody (C) include TA2650 obtained using rIL-28B (26-200) as an immunogen. Further, anti-IL-28A / B monoclonal antibody (C) can be identified by epitope competition test using TA2650.
The anti-IL-28A / B monoclonal antibody (C) binds to the epitope C. The anti-IL-28A / B monoclonal antibody (C) that binds to the epitope C has the same or highly homologous CDR. doing.

(TA2650 CDR)
The heavy chain variable region domain of TA2650 of the present invention is preferably a polypeptide (DTHMH) of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 48, 50 to A heavy chain variable region domain comprising a heavy chain complementarity determining region 2 polypeptide consisting of amino acid 66 (RIDPANGNTKFDPKFQG) and a heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 110 (DDYYGNYDAMDY). And most preferably a heavy chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 48. The light chain variable region domain is preferably a light chain complementarity determining region 1 polypeptide (RASKSVSTSGYSYMH) consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 50, 54 to 60 A light chain variable region domain comprising a polypeptide of light chain complementarity determining region 2 consisting of amino acids of (LVSNLES) and a polypeptide of light chain complementarity determining region 3 consisting of amino acids 93 to 101 (QHIRELTRS), Most preferred is a light chain variable region domain polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 50.

(Embodiment)
Although it does not limit as an IL-28B analysis method of this invention, Specifically, the following aspects can be mentioned.
Embodiments of the IL-28B analysis method of the present invention include a contact step (a) in which the first monoclonal antibody is brought into contact with a test sample, a contact step (b) in which the second antibody is brought into contact with the test sample, and a detection step. Is included. In the contact step (a) and the contact step (b), either the contact step (a) or the contact step (b) may be performed first. Therefore, there are the following two embodiments as the order of performing the contact step (a), the contact step (b), and the detection step.
(1) The contact step (a) is performed, the contact step (b) is performed, and the detection step is performed.
(2) The contact step (b) is performed, the contact step (a) is performed, and the detection step is performed.

As the IL-28B analysis method of the present invention, a sandwich method can be preferably exemplified. The sandwich method can be performed, for example, as follows.
(I) Primary reaction step The capture antibody is immobilized on an insoluble carrier such as a microplate or beads. Next, in order to prevent non-specific adsorption to the capture antibody or the insoluble carrier, the insoluble carrier is blocked with an appropriate blocking agent (for example, bovine serum albumin or gelatin). A test sample containing IL-28B is added to an insoluble carrier (microplate or bead) on which a capture antibody is immobilized, together with the primary reaction solution, and the capture antibody and IL-28B are brought into contact with each other and bound. Thereafter, antigens and contaminants not bound to the capture antibody are washed with an appropriate washing solution (for example, a phosphate buffer containing a surfactant).
(Ii) Secondary reaction step A labeled antibody (secondary antibody) labeled with an enzyme such as horseradish peroxidase (HRP) is added to the antibody that binds to IL-28B, and the labeled antibody is bound to the captured IL-28B. . By this reaction, an immunocomplex of capture antibody-IL-28B-labeled antibody is formed on an insoluble carrier (microplate or bead).
Further, instead of “labeled antibody” in which an enzyme is labeled as a secondary antibody, “biotin-labeled antibody” or “unlabeled antibody” to which biotin is bound can be used.
(Iii) Detection step An insoluble carrier (such as a microplate or a bead) is washed with a washing solution, a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody is added, and the signal is detected by reacting the enzyme with the substrate.
When a non-labeled antibody is used without directly labeling the secondary antibody, it is possible to detect the signal by labeling the antibody that binds to the secondary antibody. Furthermore, when the biotin-labeled antibody is used, a signal can be detected using enzyme-labeled avidin.

The sandwich method can be performed by enzyme immunoassay, chemiluminescence immunoassay, or radioimmunoassay. Accordingly, examples of enzymes that label antibodies include horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, and luciferase. In addition to enzymes, a luminescent substance such as an acridinium derivative, a fluorescent substance such as europium, a radioactive substance such as I ^125, or the like can be used as a labeling substance. Moreover, a substrate and a luminescence inducing substance can be appropriately selected according to the labeling substance. Furthermore, as a labeled antibody in the present invention, an antibody to which a substance that can be used for detecting a signal of an antigen-antibody reaction such as a hapten, a low molecular weight peptide, or a lectin can be used as a detection marker.

[3] IL-28B Analysis Kit The IL-28B analysis kit of the present invention is a kit that can be used in the IL-28B analysis method. IL-28B analysis kits include major and minor IL-28B analysis kits (hereinafter sometimes referred to as IL-28B analysis kits (A)) and major IL-28B analysis kits (B).

(Major and minor IL-28B analysis kit)
The major and minor IL-28B analysis kit of the present invention (IL-28B analysis kit (A)) comprises (a) an anti-IL-28B monoclonal antibody (A) or an antigen-binding fragment thereof, and (b) a SEQ ID NO: A major IL-28B composed of the amino acid sequence represented by 1; a minor IL-28B composed of the amino acid sequence represented by SEQ ID NO: 3; and IL-28A composed of the amino acid sequence represented by SEQ ID NO: 5. IL-28A / B monoclonal antibody, or an antigen-binding fragment thereof.
In the IL-28B analysis kit (A), it is possible to use an anti-IL-28B-specific monoclonal antibody instead of the anti-IL-28A / B monoclonal antibody, but preferably the anti-IL-28A / B It is a monoclonal antibody. The anti-IL-28A / B monoclonal antibody more preferably binds to major IL-28B, minor IL-28B, and IL-28A, and does not bind to a peptide fragment consisting of the amino acid sequence represented by SEQ ID NOs: 8-26. The anti-IL-28A / B monoclonal antibody (C).

(Major IL-28B analysis kit)
The major IL-28B analysis kit (IL-28B analysis kit (B)) of the present invention comprises (a) the IL-28B monoclonal antibody (B), or an antigen-binding fragment thereof, and (b) (1). The anti-IL-28B monoclonal antibody (A) or an antigen-binding fragment thereof, or (2) major IL-28B comprising the amino acid sequence represented by SEQ ID NO: 1, and minor IL comprising the amino acid sequence represented by SEQ ID NO: 3. -28B, and an anti-IL-28A / B monoclonal antibody that binds to IL-28A consisting of the amino acid sequence represented by SEQ ID NO: 5, or an antigen-binding fragment thereof.
In the IL-28B analysis kit (B), an anti-IL-28B-specific monoclonal antibody can be used instead of the anti-IL-28A / B monoclonal antibody, but preferably the anti-IL-28A / B is used. It is a monoclonal antibody. The anti-IL-28A / B monoclonal antibody more preferably binds to major IL-28B, minor IL-28B, and IL-28A, and does not bind to a peptide fragment consisting of the amino acid sequence represented by SEQ ID NOs: 8-26. The anti-IL-28A / B monoclonal antibody (C).

  The monoclonal antibody contained in the IL-28B analysis kit of the present invention may be bound to a carrier or dissolved in a buffer according to the measurement method of the analysis kit. For example, Sepharose, cellulose, agarose, dextran, polyacrylate, polystyrene, polyacrylamide, polymethacrylamide, copolymer of styrene and divinylbenzene, polyamide, polyester, polycarbonate, polyethylene oxide, hydroxypropyl methylcellulose, polyvinyl chloride, poly Mention may be made of methyl acrylate, polystyrene and polystyrene copolymers, polyvinyl alcohol, polyacrylic acid, collagen, calcium alginate, latex, polysulfone, silica, zirconia, alumina, titania, ceramics. The shape of the carrier is not particularly limited, but carriers in the form of particulate beads, plates, gels and the like can be used.

When the analysis method is an immunological method using a labeled antibody (for example, enzyme immunoassay, chemiluminescence immunoassay, fluorescent antibody method, or radioimmunoassay), the antibody is a labeling substance. In the form of a labeled antibody or a labeled antibody fragment. Specific examples of the labeling substance include peroxidase, alkaline phosphatase, β-D-galactosidase, or glucose oxidase as an enzyme, fluorescein isothiocyanate or rare earth metal chelate as a fluorescent substance, and ³ H, ¹⁴ C as a radioisotope. Or ¹²⁵ I and the like, and biotin, avidin, chemiluminescent substance, and the like. In the case of an enzyme, a chemiluminescent substance, or the like, it is not possible to provide a measurable signal by itself, and therefore it is preferable to select and include a corresponding appropriate substrate.

  The analysis kit of the present invention can contain IL-28B as a standard substance. Furthermore, the kit of the present invention may include an instruction manual that specifies that major and minor IL-28B can be specifically measured, or an instruction manual that specifies that major IL-28B can be specifically measured. it can. Such a description may be attached to the container of the analysis kit.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1: Acquisition of IL-28B specific monoclonal antibody
A solution of 0.36 mg / mL recombinant protein (rIL-28B (26-200)) of amino acid sequence 26-200 not containing signal peptide of IL-28B was mixed with an equal amount of Freund's adjuvant, and 4-6 36 μg intraperitoneally was administered to week-old BALB / c mice. Two weeks later, 20 μg of booster immunization was performed, and another 10 μg was administered into the tail vein as the final immunization.

  On the third day after the final immunization, the spleen was aseptically removed from this mouse, the spleen was loosened into individual cells using scissors and tweezers, and washed 3 times with RPMI-1640 medium. The mouse myeloma cell line NS-1 in the logarithmic growth phase was washed 3 times with RPMI-1640 medium, and the cells and spleen cells were mixed at a cell number ratio of 1:10. After centrifugation at 200 × g for 5 minutes, the supernatant is removed, 1 mL of 50% polyethylene glycol (PEG) 1500 (Roche) is slowly added while gently mixing the cell sputum, and 9 mL of RPMI-1640 medium is further added. Cell fusion.

The fused cells were removed from PEG by centrifugation (200 × g, 5 minutes), suspended in RPMI-1640 medium containing 10% fetal bovine serum and hypoxanthine, aminopterin and thymidine (HAT), and 96-well cells. Inoculated on culture plates. After culturing for 7 days and growing only the hybridoma, clones producing the antibody were searched by ELISA to obtain a hybridoma producing a monoclonal antibody having the desired reaction specificity.
The screening ELISA was performed as follows.
In a 96-well ELISA plate (Costar), rIL-28B (26-200), or a recombinant protein of the amino acid sequence 26-200 of IL-28A protein (hereinafter referred to as rIL-28A (26-200)) 50 μL of each (0.5 μg / mL) was dispensed and left overnight at 4 ° C. Next, each well of the plate was blocked with Tris-buffered saline (TBS) containing 1.6% Block Ace for 30 minutes. After removing this blocking solution, 50 μL of the hybridoma culture supernatant was added to the wells. After standing at room temperature for 60 minutes, it was washed 3 times with 0.05% Tween 20 / TBS (hereinafter referred to as TBST). Subsequently, 100 μL of horseradish peroxidase (HRP) -labeled anti-mouse IgG antibody (sheep; Paris) was added, left at room temperature for 1 hour, and then washed again with TBST four times. 50 μL of TMB substrate solution was added to each well and reacted at 25 ° C. for 30 minutes, 50 μL of a reaction stop solution was added to each well, and the absorbance at 450 nm of each well was measured.

  The obtained hybridoma was made into a single clone by the limiting dilution method, and an antibody-producing hybridoma was established. Hybridomas that produce antibodies that react to rIL-28B (26-200) but not to rIL-28A (26-200), TA2601, TA2602, TA2603, TA2613, and TA2664, and rIL-28B (26-200) and Hybridomas producing antibodies that react with rIL-28A (26-200), TA2607, TA2608, TA2611, TA2622, TA2650, TA2651 and TA2670 were obtained (hereinafter, monoclonal antibodies are also referred to by their hybridoma names). Sometimes called). As for the isotype of the obtained monoclonal antibody, TA2613 was IgG2a, and the other monoclonal antibodies were IgG1.

  The obtained hybridoma was transplanted into the abdominal cavity of a BALB / c mouse to which pristane had been intraperitoneally administered to obtain a monoclonal antibody produced in ascites. The monoclonal antibody was separated and purified by affinity chromatography using a protein G sepharose column.

  The obtained monoclonal antibody was synthesized based on the amino acid sequence (SEQ ID NO: 1) of rIL-28B Wild (major IL-28B), rIL-28B K74R (minor IL-28B) and rIL-28A, and IL-28B. Epitope analysis was performed by the ELISA method using 19 synthetic polypeptides (Table 1; SEQ ID NOs: 8 to 26) of 20 amino acids overlapping by 10 amino acids.

The results are shown in Table 2. Moreover, about TA2602, TA2664, TA2613, and TA2650, the monoclonal antibody was diluted to 1, 10, 100, or 1000 ng / mL, and ELISA was performed, and the result is shown in FIG.
Monoclonal antibodies TA2601, TA2602, and TA2603 reacted to major IL-28B, did not react to minor IL-28B and rIL-28A, and did not react to any of the 19 synthetic polypeptides. That is, TA2601, TA2602, and TA2603 were antibodies that recognize discontinuous epitopes (conformational epitopes) of major IL-28B (Table 2 and FIG. 2).
TA2613 and TA2664 reacted to major IL-28B and minor IL-28B, did not react to rIL-28A (26-200), and did not react to any of the 19 synthetic polypeptides. That is, TA2613 and TA2664 were antibodies that recognize discontinuous epitopes (conformational epitopes) of major IL-28B and minor IL-28B (Table 2).
In addition, monoclonal antibodies TA2607, TA2608, TA2611, TA2622, TA2650, TA2651 and TA2670 reacted with major IL-28B, minor IL-28B and rIL-28A, but did not react with 19 synthetic polypeptides. That is, TA2607, TA2608, TA2611, TA2622, TA2650, TA2651 and TA2670 were antibodies that recognize discontinuous epitopes (conformational epitopes) of major IL-28B, minor IL-28B and IL-28A (Table 2). ).

<Epitope competition test of monoclonal antibody>
In this example, epitope competition tests of monoclonal antibodies TA2601, TA2602, TA2603, TA2613, and TA2664 were performed.
50 μL each of rIL-28B (26-200) and (0.2 μg / mL) was dispensed into a 96-well ELISA plate (Costar) and left at 4 ° C. overnight. Next, each well of the plate was blocked with physiological saline (Saline) containing 1.6% Block Ace for 30 minutes. After removing this blocking solution, 25 μL (3.3 μg / mL) of a biotinylated monoclonal antibody and 25 μL (200 μg / mL) of the monoclonal antibody to be competed in 60.6 times the amount were added to the wells. After leaving at room temperature for 60 minutes, it was washed 3 times with 0.05% Tween 20 / Saline. Subsequently, 50 μL of horseradish peroxidase (HRP) -labeled avidin-D (Vector) was added, left at room temperature for 1 hour, and washed again with 0.05% Tween20 / Saline four times. 50 μL of TMB substrate solution was added to each well and reacted at 25 ° C. for 30 minutes, 50 μL of a reaction stop solution was added to each well, and the absorbance at 450 nm of each well was measured. The absorbance of each well to which no competing monoclonal antibody was added was taken as 100, and the absorbance was expressed as a percentage (Table 3).

TA2601, TA2602, and TA2603 compete with each other for binding to rIL-28B (26-200), and were considered to recognize the same epitope (epitope (B)). TA2613 and TA2664 were also considered to recognize the same epitope (epitope (A)).
On the other hand, in a competition test between TA2601, TA2602, and TA2603 (monoclonal antibody recognizing epitope (B)) and TA2613 and TA2664 (monoclonal antibody recognizing epitope (A)), TA2613 and TA2664 are biotinylated TA2601. , TA2602, and TA2603 were not inhibited. However, TA2601, TA2602, and TA2603 partially inhibited the binding of biotinylated TA2613 and TA2664 to rIL-28B (26-200). Therefore, it is considered that the epitope (B) and the epitope (A) are not the same, but are present at positions that influence each other.

<Determining the base sequence of the variable region gene of a monoclonal antibody>
Total RNA was extracted by a conventional method from hybridomas producing TA2601, TA2602, TA2603, TA2613, TA2664, and TA2650 antibodies, and reverse transcription was performed using oligo dT primers to prepare cDNA. From the obtained cDNA, a mouse Ig primer set (Novagen) was used to amplify the variable region gene, and PCR was performed according to the protocol. The obtained antibody variable region gene was TA cloned into the pCR2.1 vector and the nucleotide sequence was determined. The nucleotide sequence of the heavy chain variable region domain of TA2601 is shown in SEQ ID NO: 35 , the amino acid sequence is shown in SEQ ID NO: 36 , the nucleotide sequence of the light chain variable region domain is shown in SEQ ID NO: 37 , and the amino acid sequence is shown in SEQ ID NO: 38 .
The nucleotide sequence of the heavy chain variable region domain of TA2602 is shown in SEQ ID NO: 39 , the amino acid sequence is shown in SEQ ID NO: 40 , the nucleotide sequence of the light chain variable region domain is shown in SEQ ID NO: 41 , and the amino acid sequence is shown in SEQ ID NO: 42 .
The nucleotide sequence of the heavy chain variable region domain of TA2603 is shown in SEQ ID NO: 43 , the amino acid sequence is shown in SEQ ID NO: 44 , the nucleotide sequence of the light chain variable region domain is shown in SEQ ID NO: 45 , and the amino acid sequence is shown in SEQ ID NO: 46 .
The base sequence of the heavy chain variable region domain of TA2613 is shown in SEQ ID NO: 27 , the amino acid sequence is shown in SEQ ID NO: 28 , the nucleotide base sequence of the light chain variable region domain is shown in SEQ ID NO: 29 , and the amino acid sequence is shown in SEQ ID NO: 30 .
The base sequence of the heavy chain variable region domain of TA2664 is shown in SEQ ID NO: 31 , the amino acid sequence is shown in SEQ ID NO: 32 , the nucleotide sequence of the light chain variable region domain is shown in SEQ ID NO: 33 , and the amino acid sequence is shown in SEQ ID NO: 34 .
The nucleotide sequence of the heavy chain variable region domain of TA2650 is shown in SEQ ID NO: 47, the amino acid sequence is shown in SEQ ID NO: 48, the nucleotide sequence of the light chain variable region domain is shown in SEQ ID NO: 49, and the amino acid sequence is shown in SEQ ID NO: 50.

<< Comparative Example 1: Acquisition of IL-28B specific monoclonal antibody using synthetic peptide >>
As an antigen used for immunization, an IL-28B synthetic polypeptide (B26-41) (SEQ ID NO: 7) conjugated with KLH was used instead of rIL-28B (26-200). The hybridomas TA2001, TA2037, TA2042, TA2010, TA2020, TA2026, TA2044, TA2003, and TA2043 were obtained by repeating the operation of Example 1. Monoclonal antibodies TA2001, TA2037, and TA2042 reacted with synthetic peptides B26-41 and rIL-28B (26-200), but hardly reacted with rIL-28A (26-200). That is, TA2001, TA2037, and TA2042 were antibodies that recognize a continuous epitope of IL-28B (Table 4). Monoclonal antibodies TA2010, TA2020, TA2026, TA2044, TA2003, and TA2043 reacted with synthetic peptides B26-41, rIL-28B (26-200), and rIL-28A (26-200). TA2010, TA2020, TA2026, TA2044, TA2003, and TA2043 were antibodies that recognize continuous epitopes of IL-28A and IL-28B (Table 4).

Example 4: Measurement method specific to major and minor IL-28B
Monoclonal antibody TA2650 was diluted with 10 mM HEPES buffer (pH 7.4: hereinafter referred to as HBS buffer) containing 150 mM NaCl to a final concentration of 2 μg / mL, and 96 well microplate (manufactured by NUNK) per well. Dispense 100 μL each. After standing at 4 ° C. overnight, the plate was washed twice with 400 μL of HBS buffer, added with 400 μL of HBS buffer containing 0.1% casein-Na (hereinafter referred to as blocking solution), and further at room temperature. Let stand for 30 minutes.

  After removing the blocking solution, the plate was washed twice with 400 μL of an HBS buffer solution (hereinafter referred to as a washing solution) containing 0.05% Tween20. To 50 μL of HBS buffer (hereinafter referred to as dilution buffer) containing 0.1% casein-Na, 1% mouse serum, 1% BSA, and 0.05% Tween20, rIL-28BW (HeLa) (major IL- 28B), rIL-28B W (Invitrogen) (major IL-28B), rIL-28B K74R (HeLa) (minor IL-28B), rIL-28B K74R (R & D) (minor IL-28B), rIL-28A (R & D) ) And rIL-29 (R & D) were diluted to 0 to 10 ng / mL with a dilution buffer, added in 50 μL each well, and allowed to react at room temperature for 1 hour with stirring. Wash with 400 μL of washing solution 3 times, and further add alkaline phosphatase (AP) -labeled monoclonal antibody TA2664 Fab ′ fragment to 30 ng / mL with dilution buffer, add 100 μL to each well, and stir at room temperature for 1 hour. I let you. After the reaction, the plate was washed 5 times with 400 μL of the washing solution, added with 50 μL of a substrate (CDP-star with Sappire II) solution, reacted at room temperature in the dark for 20 minutes, and then luminescence was measured with a SpectraMax plate reader.

  As shown in FIG. 3, major IL-28B and minor IL-28B could be specifically measured with TA2650 and TA2664. Moreover, a wide dynamic range of 0.1 pg / mL to 10,000 pg / mL was obtained. The minimum detection limit (sensitivity) was 0.1 pg / mL.

<< Example 5: Measurement method specific to major and minor IL-28B >>
The procedure of Example 4 was repeated except that TA2664 was used instead of TA2650 as the solid phase antibody and TA2650 was used instead of TA2664 as the labeled antibody.

  As shown in FIG. 4, major IL-28B and minor IL-28B could be specifically measured even when TA2650 and TA2664 were replaced with solid phase antibodies and labeled antibodies. Moreover, a wide dynamic range of 0.1 pg / mL to 10,000 pg / mL was obtained. The minimum detection limit (sensitivity) was 0.1 pg / mL.

<< Example 6: Measurement method specific to major IL-28B >>
The procedure of Example 4 was repeated except that TA2602 was used instead of TA2650 as the solid phase antibody.

  As shown in FIG. 5, major IL-28B could be specifically measured with TA2602 and TA2664. Moreover, a wide dynamic range of 0.1 pg / mL to 10,000 pg / mL was obtained. The minimum detection limit (sensitivity) was 0.1 pg / mL.

<< Example 7: Measurement method specific to major IL-28B >>
The procedure of Example 6 was repeated except that TA2650 was used instead of TA2664 as the labeled antibody.

  As shown in FIG. 6, the major IL-28B could be specifically measured with TA2602 and TA2650. Moreover, a wide dynamic range of 0.1 pg / mL to 10,000 pg / mL was obtained. The minimum detection limit (sensitivity) was 0.1 pg / mL.

<< Comparative Example 2 >>
In this comparative example, rIL-28B W (Invitrogen) (major IL-28B), rIL-28B K74R (R & D) (minor IL-28B), a kit using an IL-28B specific ELISA kit manufactured by R & D. The attached rIL-28B (DuoSet) (minor IL-28B), rIL-28A (R & D), and rIL-29 (R & D) were measured. The measurement method was performed according to the instructions. The results are shown in FIG. Major IL-28B and minor IL-28B can be measured, but it was about 100 pg / mL.

<< Example 8: Measurement of IL-28B in patients with hepatitis C >>
In this example, the measurement method of Example 6 was used to measure IL-28B of 32 samples of serum or plasma from hepatitis C patients. The results are shown in Table 5. IL-28B in the serum of hepatitis C patients was often 1 pg / mL, but IL-28B could be detected in 31 of 32 samples.

  According to the method for predicting therapeutic effect of a drug in a hepatitis C patient of the present invention, for example, the therapeutic effect of combined administration of PEGylated IFN and ribavirin can be accurately predicted. By predicting the therapeutic effect of HCV, it is possible to reduce the treatment cost.

Claims (18)

It binds to major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1 and minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3, and to IL-28A consisting of the amino acid sequence represented by SEQ ID NO: 5. An anti-IL-28B monoclonal antibody, or an antigen-binding fragment thereof, which does not bind and does not bind to a peptide fragment consisting of the amino acid sequence represented by SEQ ID NOs: 8-26,
(1) Polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 28, and heavy chain complementarity determining region 2 consisting of amino acids 50 to 66 A heavy chain variable region domain comprising a polypeptide and a heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 113, and amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 30; The light chain complementarity determining region 1 polypeptide, the light chain complementarity determining region 2 polypeptide consisting of amino acids 54 to 60, and the light chain complementarity determining region 3 consisting of amino acids 93 to 101 A light chain variable region domain comprising a polypeptide; or (2) heavy chain complementation determination comprising amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 32 A heavy chain comprising a polypeptide of region 1, a polypeptide of heavy chain complementarity determining region 2 consisting of amino acids 50 to 66, and a polypeptide of heavy chain complementarity determining region 3 consisting of amino acids 99 to 109 A variable region domain; a polypeptide of light chain complementarity determining region 1 consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 34; and a light chain complementarity determining region consisting of amino acids 54 to 60 It makes the chromophore at the distal end monoclonal antibodies having a light chain variable region domain comprising two polypeptide, and a light chain complementarity determining region 3 of the polypeptide consisting of 93 th to 101 th amino acid, or an antigen-binding fragment thereof.   A monoclonal antibody, or an antigen-binding fragment thereof, that competes with the monoclonal antibody of claim 1 for binding of major IL-28B and minor IL-28B.   It binds to major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1, and to minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3 and IL-28A consisting of the amino acid sequence represented by SEQ ID NO: 5. An anti-IL-28B monoclonal antibody, or an antigen-binding fragment thereof, which does not bind and does not bind to a peptide fragment consisting of the amino acid sequence represented by SEQ ID NOs: 8-26. (1) A heavy chain complementarity determining region 1 polypeptide consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 36, and a heavy chain complementarity determining region 2 consisting of amino acids 50 to 66 A polypeptide, a heavy chain variable region domain comprising the heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 106, and amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 38 Polypeptide of light chain complementarity determining region 1, polypeptide of light chain complementarity determining region 2 consisting of amino acids 54 to 60, polypeptide of light chain complementarity determining region 3 consisting of amino acids 93 to 101 A light chain variable region domain comprising:
(2) Polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 40, heavy chain complementarity determining region 2 consisting of amino acids 50 to 66 A polypeptide, a heavy chain variable region domain comprising the heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 106, and amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 42 Polypeptide of light chain complementarity determining region 1, polypeptide of light chain complementarity determining region 2 consisting of amino acids 54 to 60, polypeptide of light chain complementarity determining region 3 consisting of amino acids 93 to 101 Or (3) a heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 44 A heavy chain variable region domain comprising a heavy chain complementarity determining region 2 polypeptide consisting of amino acids 50 to 66, a heavy chain complementarity determining region 3 polypeptide consisting of amino acids 99 to 106, and A polypeptide of the light chain complementarity determining region 1 consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 46; a polypeptide of the light chain complementarity determining region 2 consisting of amino acids 54 to 60; The monoclonal antibody or antigen-binding fragment thereof according to claim 3, which has a light chain variable region domain comprising the light chain complementarity determining region 3 polypeptide consisting of amino acids 93 to 101.   A monoclonal antibody, or an antigen-binding fragment thereof, that competes with the monoclonal antibody according to claim 4 for binding to major IL-28B.   The hybridoma which produces the monoclonal antibody as described in any one of Claims 1-5.   A major and minor IL-28B assay comprising the step of contacting the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 with a test sample that may contain IL-28B. Method. (1) contacting the monoclonal antibody according to claim 1 or 2 or an antigen-binding fragment thereof with a test sample that may contain IL-28B; and (2) a second that binds to IL-28B. Contacting the antibody with a test sample;
The major and minor IL-28B analysis method according to claim 7. A second antibody that binds to IL-28B,
It binds to major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1, minor IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 3, and IL-28A consisting of the amino acid sequence represented by SEQ ID NO: 5. The major and minor IL-28B analysis method according to claim 8, which is an anti-IL-28A / B monoclonal antibody, or an antigen-binding fragment thereof. The anti-IL-28A / B monoclonal antibody is
A polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 48; a polypeptide of heavy chain complementarity determining region 2 consisting of amino acids 50 to 66; Heavy chain variable region domain comprising heavy chain complementarity determining region 3 consisting of amino acids 99 to 110, and light chain complementarity determining region consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 50 1 polypeptide, light chain complementarity determining region 2 polypeptide consisting of amino acids 54 to 60, and light chain variable region comprising a light chain complementarity determining region 3 polypeptide consisting of amino acids 93 to 101 domain,
The method for major and minor IL-28B analysis according to claim 9, which is a monoclonal antibody having   Contacting the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 3 to 5 with a test sample that may contain IL-28B. -28B analysis method. (1) contacting the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 3 to 5 with a test sample that may contain IL-28B; and (2) IL-28B Contacting the test sample with a second antibody that binds to
The major IL-28B analysis method according to claim 11, comprising: A second antibody that binds to IL-28B,
(1) The monoclonal antibody according to claim 1 or 2, or an antigen-binding fragment thereof, or (2) Major IL-28B consisting of the amino acid sequence represented by SEQ ID NO: 1, represented by SEQ ID NO: 3 An anti-IL-28A / B monoclonal antibody that binds to a minor IL-28B consisting of an amino acid sequence and IL-28A consisting of an amino acid sequence represented by SEQ ID NO: 5, or an antigen-binding fragment thereof.
The major IL-28B analysis method according to claim 12. The anti-IL-28A / B monoclonal antibody is
A polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 48; a polypeptide of heavy chain complementarity determining region 2 consisting of amino acids 50 to 66; Heavy chain variable region domain comprising heavy chain complementarity determining region 3 consisting of amino acids 99 to 110, and light chain complementarity determining region consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 50 1 polypeptide, light chain complementarity determining region 2 polypeptide consisting of amino acids 54 to 60, and light chain variable region comprising a light chain complementarity determining region 3 polypeptide consisting of amino acids 93 to 101 domain,
The major IL-28B analysis method according to claim 13, which is a monoclonal antibody having (A) the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, and (b) a major IL-28B comprising the amino acid sequence represented by SEQ ID NO: 1, and the amino acid sequence represented by SEQ ID NO: 3. An anti-IL-28A / B monoclonal antibody, or an antigen-binding fragment thereof, which binds to IL-28A consisting of the minor IL-28B and the amino acid sequence represented by SEQ ID NO: 5,
Major and minor IL-28B analysis kits. The anti-IL-28A / B monoclonal antibody is
A polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 48; a polypeptide of heavy chain complementarity determining region 2 consisting of amino acids 50 to 66; Heavy chain variable region domain comprising heavy chain complementarity determining region 3 consisting of amino acids 99 to 110, and light chain complementarity determining region consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 50 1 polypeptide, light chain complementarity determining region 2 polypeptide consisting of amino acids 54 to 60, and light chain variable region comprising a light chain complementarity determining region 3 polypeptide consisting of amino acids 93 to 101 domain,
The major and minor IL-28B analysis kit according to claim 15, which is a monoclonal antibody having (A) the monoclonal antibody according to any one of claims 3 to 5, or an antigen-binding fragment thereof, and (b) (1) the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, Or (2) major IL-28B comprising the amino acid sequence represented by SEQ ID NO: 1, minor IL-28B comprising the amino acid sequence represented by SEQ ID NO: 3, and IL- comprising the amino acid sequence represented by SEQ ID NO: 5. An anti-IL-28A / B monoclonal antibody that binds to 28A, or an antigen-binding fragment thereof,
A kit for analysis of major IL-28B. The anti-IL-28A / B monoclonal antibody is
A polypeptide of heavy chain complementarity determining region 1 consisting of amino acids 31 to 35 in the amino acid sequence represented by SEQ ID NO: 48; a polypeptide of heavy chain complementarity determining region 2 consisting of amino acids 50 to 66; Heavy chain variable region domain comprising heavy chain complementarity determining region 3 consisting of amino acids 99 to 110, and light chain complementarity determining region consisting of amino acids 24 to 38 in the amino acid sequence represented by SEQ ID NO: 50 1 polypeptide, light chain complementarity determining region 2 polypeptide consisting of amino acids 54 to 60, and light chain variable region comprising a light chain complementarity determining region 3 polypeptide consisting of amino acids 93 to 101 domain,
The kit for major IL-28B analysis according to claim 17, which is a monoclonal antibody having

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