JP5920761B2 - Anti-DCD monoclonal antibody - Google Patents

Description

  The present invention relates to a monoclonal antibody that recognizes human darmsidin (hereinafter, sometimes referred to simply as DCD), a hybrid cell that produces the monoclonal antibody, a method for producing the monoclonal antibody, and a method of using the monoclonal antibody.

  DCD is an antibacterial peptide consisting of 110 amino acids isolated from human sweat by Birgit Shitek et al. (Patent Document 1, Non-Patent Document 1). It has been confirmed that it exhibits antibacterial activity against Escherichia coli, enterococci, Staphylococcus aureus, and Candida under the same pH and the same salt concentration as human sweat, and it is secreted constantly into human sweat. Therefore, compared to other antibacterial peptides (β-defensin, cathelicidin, etc.) that are expressed in the skin during inflammation, it is more likely to control infection of skin microorganisms, and development of skin diseases involving microorganisms and viruses It is speculated that it is strongly related to.

  In addition, it has been reported that the amount of DCD in sweat of patients with atopic dermatitis is smaller than that of healthy individuals (Non-patent Document 2), and may be related to deterioration of skin symptoms due to Staphylococcus aureus in atopic dermatitis Sex has been suggested. Furthermore, it has been reported that several types of DCD degradable peptides exist in sweat, and there are individual differences in the abundance ratio of degradable DCD (Non-patent Document 3). It is also possible that there is an influence on individual differences in the susceptibility to infection. Therefore, if the degree of contribution of DCD to the disease becomes clear, the infectivity of microorganisms to the skin is evaluated by measuring the total amount of DCD or the amount of decomposed DCD in biological samples such as sweat. There is a possibility that it can be utilized in the prevention and treatment of cancer.

  Until now, the SELDI-TOF-MS method has been frequently used for the measurement of DCD in a biological sample, but this method has been difficult to obtain an accurate concentration. As a method for measuring the amount of DCD in a biological sample, an immunological measurement method using an antibody that recognizes an amino acid sequence specific for DCD is simple and accurate. Examples of immunological measurement methods include enzyme immunoassay and a method for examining many parameters at once with high sensitivity using a plurality of beads to which antibodies are bound (Bio-Plex suspension array system (manufactured by BioRad)). Can be exemplified). In particular, the latter is an excellent method capable of examining a large number of parameters with a small amount of specimen, and it is also possible to examine the abundance ratio of degraded DCD using a plurality of antibodies having different antigen recognition properties. It becomes.

  As anti-DCD antibodies, many polyclonal antibodies are commercially available in the past. Polyclonal antibodies may cause problems in specificity in antigen recognition, and monoclonal antibodies are preferred for more accurate DCD quantification. As a commercially available monoclonal antibody, an IgM class anti-DCD monoclonal antibody (G-81) produced by Sagawa et al. Of Wakayama Medical University is known (Non-patent Document 4). However, the IgG class antibody is generally more specific than the IgM class in terms of specificity, and the antigen aggregation and complement activation strength is higher in the IgM class antibody than in the IgG class. For the reason that DCD is more accurately detected, an IgG class antibody is preferable because it is stronger. In particular, the IgG class antibody is suitable for the above-described method of analyzing a large number of parameters using a plurality of types of antibodies and beads, and the performance and advantages of this method are completely eliminated with an IgM class antibody. There is a problem that it cannot be utilized. Here, as an IgG class monoclonal antibody, we already have a hybridoma 10C-3 that produces an anti-DCD monoclonal antibody (10C-3 antibody) having a recognition epitope in the 92nd to 108th amino acid sequence of the full-length DCD. A strain (FERM P-21792) is produced (Patent Document 2). However, some DCD-degraded peptides present in sweat do not have the amino acid sequence recognized by the 10C-3 antibody. There was a problem that it could not be detected.

U.S. Pat. No. 7,348,409 Japanese Patent Laid-Open No. 10-241731

Birgit Shittek et al. Nature Immunology, 2001, 2 (12): 1133-1137. Siegbert Rieg et al. J. et al. Invest. Dermatol. , 2006, 126: 354-365. Siegbert Rieg et al. J. et al. Immunol. , 2005, 174: 8003-8010. Kazunori Sagawa et al. Int. J. et al. Legal. Med. , 2003, 117: 90-95.

  An object is to provide an IgG class monoclonal antibody that recognizes DCD and a hybridoma that produces the monoclonal antibody. Another object of the present invention is to provide a method for examining DCD in a biological sample using this antibody.

  As a result of intensive studies to solve the above problems, the present inventors synthesized a peptide having the 63rd to 81st sequences among the amino acid sequences of full-length human DCD, and used it as a keyhole limpet as a carrier protein. The antibody of the present invention could be obtained by using a KLH-added DCD peptide obtained by adding hemocyanin (KLH) as an antigen. That is, antibody-producing cells obtained from an animal immunized with the antigen and myeloma cells were fused by a cell fusion method, and a hybridoma capable of producing an IgG class monoclonal antibody that recognizes human DCD by screening was obtained. Then, by culturing this hybridoma, it was found that an IgG class monoclonal antibody that recognizes human DCD can be produced, and the present invention has been completed.

That is, the present invention relates to the following monoclonal antibodies (1) to (10).
(1) An IgG class anti-DCD monoclonal antibody that reacts with a peptide having the amino acid sequence shown in SEQ ID NO: 7 and SEQ ID NO: 6 in the sequence listing.
(2) The monoclonal antibody according to (1) above, which recognizes the 66th to 81st regions in the amino acid sequence of DCD.
(3) The monoclonal antibody according to (1), which is obtained by immunizing a non-human animal with a KLH-added DCD peptide obtained by adding KLH to a peptide having the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing as an antigen.
(4) The monoclonal antibody according to (1), which is produced by the 6B-10 strain (FERMP P- 22134), which is a hybridoma.
(5) 6B-10 strain (FERM P- 22134) which is a hybridoma.
(6) A method for producing an IgG class anti-DCD monoclonal antibody comprising the step of growing the hybridoma according to (5).
(7) A method for detecting DCD, comprising the step of mixing a sample with the monoclonal antibody according to any one of (1) to (4).
(8) The detection method according to (7), wherein the sample is derived from human sweat.
(9) A DCD detection kit comprising the monoclonal antibody according to any one of (1) to (4) or the labeled monoclonal antibody.

  According to the present invention, it is possible to provide an IgG class monoclonal antibody capable of detecting DCD with high sensitivity. The monoclonal antibody provided by the present invention can be used for measuring the amount of DCD contained in a biological sample such as sweat.

It is the figure which confirmed the antigen recognition specificity of the anti- DCD monoclonal antibody (6B-10 antibody) (Example). It is the figure which confirmed and showed the reactivity with anti- DCD monoclonal antibody (6B-10 antibody) to DCD in sweat (Example). It is the figure which showed the analytical curve in the quantification method of DCD using an anti- DCD monoclonal antibody (6B-10 antibody) (Example). It is the figure which showed the difference in the detection amount of 6B-10 antibody and 10C-3 antibody in DCD determination contained in sweat.

The monoclonal antibody of the present invention is any IgG class monoclonal antibody that recognizes DCD, and any monoclonal antibody that reacts with a peptide consisting of the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 6 in the sequence listing. May be.
Further, it is desirable that the antibody recognizes the 66th to 81st region in the amino acid sequence of DCD.
Furthermore, the monoclonal antibody of the present invention includes not only the whole antibody molecule but also an antibody fragment capable of recognizing the specific region of DCD.
Here, to recognize a specific region of DCD means to bind to the specific region of DCD or to perform some action by binding.
Examples of the DCD include human DCD, chimpanzee DCD, orangutan DCD-like protein, rhesus monkey DCD-like protein, and common marset DCD-like protein. Among these, human DCD is preferable.
As such a monoclonal antibody, a monoclonal antibody that reacts with each peptide consisting of the amino acid sequences shown in SEQ ID NOs: 2, 3, 4, 5 and 6 in the sequence listing, and 6B-10 strain (FERM P- 22134) are produced. A monoclonal antibody etc. are mentioned. Among these, the monoclonal antibody produced by the 6B-10 strain (FERMP P- 22134) is preferable. Since DCD does not have a three-dimensional structure, has low antigen recognition ability, and it is difficult to obtain a monoclonal antibody, the “monoclonal antibody” of the present invention is very useful.

The hybridoma of the present invention is an IgG class monoclonal antibody that recognizes DCD and can produce a monoclonal antibody that reacts with a peptide consisting of the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 6 in the sequence listing. Any hybridoma may be used. Examples of such hybridomas include 6B-10 strain (FERM P- 22134).
The hybridoma of the present invention comprises a spleen cell excised from the spleen of a mouse, a rat or the like whose antibody titer in serum is sufficiently increased by using a peptide having the amino acid sequence from 63rd to 81st of human DCD as an immunizing antigen. It is preferably obtained by fusing myeloma cells and culturing the fused cells in a HAT medium or the like. In order to obtain an IgG class monoclonal antibody that recognizes human DCD, it is preferable to immunize the mouse, rat, etc. for a period during which the antibody titer in the serum can be sufficiently increased.
Examples of the immunizing antigen for obtaining a hybridoma include human DCD, His-tag fusion human DCD, a peptide having a part of the amino acid sequence of human DCD, a carrier protein-added protein thereof, and the like. Among these, a KLH-added DCD peptide obtained by adding KLH to a peptide having the 63rd to 81st amino acid sequence of human DCD is preferable as an immunizing antigen.
Moreover, since the immunogenicity (antigenicity) of DCD is very weak, a hybridoma producing an IgG antibody cannot usually be obtained by cloning only a number. Therefore, in order to obtain a DCD-recognizing IgG antibody-producing hybridoma, the screening of the hybridoma is not carried out generally for about 1,000 clones, but at least 10,000 clones, preferably 100,000 clones or more. Need to be screened.

The “monoclonal antibody production method” of the present invention includes any production method as long as it uses a hybridoma capable of producing an IgG class monoclonal antibody that recognizes DCD.
For example, a method in which a hybridoma capable of producing an IgG class monoclonal antibody that recognizes DCD is grown in a liquid medium or in the abdominal cavity of a non-human animal to produce and accumulate the monoclonal antibody, and a method for collecting the monoclonal antibody can be mentioned. . Examples of animals other than humans include animal species that do not immunologically exclude hybridomas, such as mice, nude mice, nude rats, etc. that are syngeneic with myeloma cells.

The DCD detection method of the present invention may be any method as long as it uses a IgG class monoclonal antibody that recognizes DCD.
For example, in the detection method of the present invention, DCD contained in a sample such as sweat can be detected and quantified by performing an enzyme immunoassay using an IgG class monoclonal antibody that recognizes DCD.
In addition, a method for inspecting many parameters at a time with high sensitivity using a plurality of beads combined with an IgG class monoclonal antibody that recognizes DCD of the present invention and another antibody (Bio) -Plex suspension array system (manufactured by BioRad, etc.) and the like can also be performed. In this method, since a plurality of antibodies having different antigen recognizing properties are used, the abundance ratio of each cleaved form of DCD can be examined, and individual differences in antibacterial activity can be examined. Examples of such a combination of a plurality of antibodies include a combination of 6B-10 antibody and 10C-3 antibody, which are combinations of the same IgG classes, and a combination of IgG class and IgM class includes 6B- The combination of 10 antibody and G-81 antibody etc. are mentioned. Moreover, the combination of 3 types of antibodies, 6B-10 antibody, 10C-3 antibody, and G-81 antibody is also mentioned.

The detection kit of the present invention refers to a kit for examining DCD, and may be any kit as long as it uses an IgG class monoclonal antibody that recognizes DCD. For example, a kit using a combination of an IgG class monoclonal antibody that recognizes DCD and a purified human DCD that is a standard sample can be used. The monoclonal antibody contained in this kit is preferably a labeled antibody for detection and quantification of DCD. For example, an antibody labeled with alkaline phosphatase or the like can be mentioned. Any known labeling substance for this antibody can be used.
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.

Method for producing DCD monoclonal antibody Preparation of antigen protein As an immunizing antigen, a peptide (hereinafter referred to as DCD peptide) having the 63rd to 81st sequence (SSLLEKGLDGAKKAVGGLGLGLG: Sequence Listing SEQ ID NO: 7) of the amino acid sequence of full-length human DCD is prepared by synthesis, and carrier protein A protein to which keyhole limpet hemocyanin (KLH) was added was used.
The addition of KLH was performed using the maleimide method with cysteine added to the C-terminal of the DCD peptide. The synthesis of the above KLH-added DCD peptide was performed by requesting Sigma Aldrich.

2. Preparation of hybridoma (1) Immunization A PBS solution of the antigen prepared in step 1 (DCD peptide concentration excluding KLH: 3.0 mg / ml) and TiterMax Gold (manufactured by CytRx), which is an adjuvant, are mixed in equal amounts, and mice (BALB) are in W / O micelle state (/ C, 7 weeks old) was administered subcutaneously to the back (dose: 150 μg / mouse as the amount of DCD peptide excluding KLH). Further, similarly, an antigen mixed with TiterMax Gold was boosted twice at intervals of 10 to 14 days. Two weeks after the last booster, the same amount of antigen was mixed in a PBS solution and injected intraperitoneally for final immunization. Spleen cells were removed 3 days after the final immunization.
The antibody titer was determined by collecting blood from the mouse inferior vein plexus 7 days after the booster immunization and measuring the antibody titer in the serum by the following enzyme immunoassay to confirm that the antibody against the antigen was produced. did.

(2) Confirmation of antibody titer The antibody titer was confirmed by enzyme immunoassay using a KLH-added DCD peptide as a solid phase. First, using a coating buffer (carbonate-bicarbonate buffer, pH 10), the KLH-added DCD peptide was diluted to 10 μg / ml, and dispensed into a 96-well EIA / RIA plate (Costar, High Binding) (50 μL / well). And physisorbed by incubating at 4 ° C. overnight (about 14 hours). The plate was washed 3 times with PBS containing 0.1% Tween 20 (hereinafter abbreviated as PBST), and then a blocking solution (PBS containing 0.5% BSA) was added at 100 μL / well and blocked at room temperature for 2 hours.

After removal of the blocking solution, serial dilutions of serum (50 μl / well) obtained from mice as a primary antibody with PBS were added and incubated at room temperature for 2 hours. After the plate was washed 4 times with PBST, a peroxidase-labeled anti-mouse Ig (G + M) goat polyclonal antibody (manufactured by Tago Products) was reacted as a secondary antibody for 1 hour at room temperature, and then ortho-phenylenediamine (manufactured by Sigma) was included. Color was developed by adding a peroxidase substrate solution. Ten minutes later, 1.5N sulfuric acid was added to stop color development, and then the absorbance at 492 nm was measured with a microplate reader.
In the immunized mice in this example, a difference in absorbance was observed as compared to serum obtained from naive mice. Absorbance measured using 1000-fold diluted serum was 0.05 or less in naïve mice, but the serum of immunized mice was 2.0, confirming that the antibody titer had increased It was done. Mice with sufficiently increased antibody titers were used in the next step as a source of antibody-producing cells.

(3) Screening of cell fusion and hybridoma Spleen cells extracted from the spleen of a mouse with a sufficiently high antibody titer and mouse myeloma cells (FO) (ATCC) were mixed at a ratio of 5 to 1, and 50% polyethylene glycol Cell fusion was carried out in the presence of a molecular weight of 1400 to 1600 (manufactured by Sigma).
The fused cells are suspended in HAT medium (RPMI medium containing hypoxanthine, aminopterin and thymidine) so as to be 2 × 10 ⁶ / mL as spleen cells, and 96-well culture plates (CORNING, Inc.) on which feeder cells have been seeded 0.1 mL each. This was cultured at 37 ° C. in a 5% CO ₂ incubator, and after about 2 weeks, the culture supernatant of the well in which the hybridoma had grown was subjected to KLH in the enzyme immunoassay shown in the above antibody titer measurement. By experiments in which the added DCD peptide and KLH and the primary antibody were replaced with the culture supernatant, a strain that responded to the KLH-added DCD peptide and was promising to produce an antibody that did not react with KLH was selected. Since DCD does not have a three-dimensional structure and has poor antigen recognizability, screening of 1,000 clones does not yield the desired monoclonal antibody, screening was performed with 10,000 clones, and 8 types of anti-DCD monoclonal antibody-producing hybridomas were produced. Established. Of the 8 species, 5 were of the IgM class and 3 were of the IgG class. Among the IgG class hybridomas, one strain that produces an antibody that is highly reactive with the antigen has been designated by the applicant as an independent administrative agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1 East 1st, Tsukuba, Ibaraki, Japan). The deposit procedure was performed on June 23, 2011 at the address No. 1 (Chuo 6), and a receipt number (FERM P- 22134) was assigned. Hereinafter, the anti-DCD monoclonal antibody produced by this hybridoma 6B-10 strain (FERM P- 22134) is referred to as 6B-10 antibody.

3. Production of monoclonal antibody Two weeks before the administration of the hybridoma, an 8 week-old female BALB / C mouse that had been injected intraperitoneally with 0.5 mL of pristane was treated with the hybridoma obtained above with 5 × 10 ⁶ cells. The dose was administered intraperitoneally. About 10 days after the administration of the hybridoma, ascites was collected and centrifuged to obtain a supernatant. The supernatant was mixed with an equal amount of an adsorption buffer (3 mol / L NaCl-1.5 mol / L Glycine-NaOH, pH 8.5) and then filtered. This filtrate is passed through a protein A column (manufactured by Pharmacia) equilibrated with an adsorption buffer solution, and the antibody is adsorbed onto the column, and then eluted from the column with a 0.1 mol / L citrate buffer solution (pH 3.0). Anti-DCD monoclonal antibody (6B-10 antibody) was purified.

4). Identification of immunoglobulin class and subclass of anti-DCD monoclonal antibody (6B-10 antibody) The immunoglobulin class of the 6B-10 antibody prepared in (1) was confirmed using the Mouse Monoclonal Antibody Isotyping Test Kit (manufactured by Serotec).
The 6B-10 antibody was diluted to 1.0 μg / ml using PBS containing 0.1% BSA, added to the tube attached to the kit, and the reagent in the tube was dissolved. Immediately, the strip attached to the kit was immersed in the lysis solution in the tube, and after 10 minutes, the position of the band appearing on the strip was observed to determine the immunoglobulin class. As a result, it was confirmed that the immunoglobulin class of the 6B-10 antibody was the IgG2a) and κ light chain desired in the present invention.

  Hereinafter, the 6B-10 antibody obtained by the present invention and the IgG class anti-DCD mouse monoclonal antibody (10C-3 antibody (FERM P-21792)) or IgM class anti-DCD mouse monoclonal antibody obtained as conventional techniques (G-81 antibody) was compared, and the antigen recognition specificity of 6B-10 antibody was confirmed.

5. 2. Confirmation of antigen recognition specificity of anti-DCD monoclonal antibody (6B-10 antibody) Western blot analysis was performed to confirm the specificity of antigen recognition of the 6B-10 antibody prepared in (1). As antigens for Western blotting, approximately 16 kDa polyhistidine (hereinafter abbreviated as His-tag) and DCD protein (Full-DCD; having 20th to 110th amino acid sequence of full-length DCD) expressed recombinantly in E. coli. A fusion protein was used.
A general technique was used as the recombinant expression technique. That is, reverse transcriptase reaction using random primers (manufactured by GE Healthcare Science) was performed on total RNA extracted from human skin tissue to prepare cDNA.
The obtained cDNA was inserted into pGEM T Easy Vector (Promega), which is a commercially available plasmid vector, and stabilized by TA cloning. Using this DCD-inserted pGEM T Easy Vector as a template, a sequence obtained by removing the signal sequence region from the cDNA sequence of human DCD (SEQ ID NO: 1) was amplified by the PCR method, and a commercially available plasmid vector pET28 (manufactured by Novagen) ) To create a DCD / pET28 plasmid vector.
This DCD / pET28 plasmid vector was transformed into Escherichia coli (BL21 (DE3) strain), and colonies were formed on a kanamycin-containing agar medium. One colony was collected and cultured with shaking in a kanamycin-containing medium, and expression of the recombinant protein was induced by adding isopropyl-β-thiogalactopyranoside in the logarithmic growth phase.
The spun E. coli was lysed with Tris-HCl buffer containing guanidine hydrochloride. The lysate was purified by immobilized metal affinity chromatography using Ni-NTA agarose (manufactured by QIAGEN), followed by gradual lowering of the guanidine concentration by dialysis to obtain a polyhistidine fusion protein. Here, since it was confirmed that a small amount of protein having a molecular weight different from about 16 kDa corresponding to the target His-tag fusion DCD was mixed, polyacrylamide gel electrophoresis was performed, and the band of the target molecular weight was cut out from the gel. The protein was eluted from the gel using an electroeluter (BioRad).
The obtained His-tag fusion DCD protein was developed on SDS-PAGE under reducing conditions, transferred to a PVDF membrane, and 4 times with TBST containing 0.1% BSA (TBS containing 0.05% Tween 20, pH 7.4). After blocking overnight at 0 ° C., 4000-fold diluted solution of 6B-10 antibody (3.3 mg / ml) or 4000-fold diluted solution of 10C-3 antibody (3.1 mg / ml) as primary antibody, alkaline phosphatase as secondary antibody A labeled anti-mouse IgG antibody (manufactured by CAPPEL) was reacted. After the PVDF membrane was washed with TBST, a mixed solution of NBT (nitroblue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3-indolyl phosphate) was added as a substrate for color development.
As a result, it was confirmed that the 6B-10 antibody reacted with the His-tag fusion DCD protein in the same manner as the 10C-3 antibody (FIG. 1). In FIG. 1, lane M represents the molecular weight marker, lane A represents the Coomassie blue (CBB) staining result on the PVDF membrane of the His-tag fusion DCD protein, lane B represents the 6B-10 antibody, and lane C represents the 10C- The result of the Western blot analysis by 3 antibodies is represented.

6). Identification of recognition epitope of anti-DCD monoclonal antibody (6B-10 antibody) In order to confirm the recognition epitope of the 6B-10 antibody prepared in Step 1, the peptide (non-patent document 3) known to exist in sweat as a degraded form (cleaved form) of DCD is shown in Table 1 below. Synthetic peptides consisting of the amino acid sequences shown (SEQ ID NO: 2 to 6) were prepared (manufactured by AnyGen), and the reactivity of 6B-10 antibody was confirmed by enzyme immunoassay using these as antigens.

The enzyme immunoassay was performed by the following method.
First, each synthetic peptide was diluted to 100 pmol / ml with -PBS (-), dispensed into a 96-well EIA / RIA plate (Costar, High Binding) (50 μL / well), and incubated at room temperature for about 18 hours. To physically adsorb.
After washing the plate 10 times with sterile distilled water, 100 μL / well of blocking solution (PBS containing 1% BSA) was added and blocked at 37 ° C. for 1 hour. After removing the blocking solution and washing 10 times with sterilized distilled water, 8000-fold diluted solution of 6B-10 antibody (3.3 mg / ml) as the primary antibody and 8000-fold diluted solution of 10C-3 antibody (3.1 mg / ml) Alternatively, G-81 antibody (50 μl / well) was dispensed and incubated at 4 ° C. for about 18 hours.
After the plate was washed 10 times with sterile distilled water, a peroxidase-labeled anti-mouse Ig (G + M) goat polyclonal antibody (manufactured by Tago Products) diluted as a secondary antibody was reacted at 37 ° C. for 1 hour, and then orthophenylenediamine (Sigma). Peroxidase substrate solution containing the product was added to cause color development. Ten minutes later, 1.5N sulfuric acid was added to stop color development, and then the absorbance at 492 nm was measured with a microplate reader.
As a result, the reactivity of the 6B-10 antibody, 10C-3 antibody and G-81 antibody to the synthetic peptides was as shown in Table 2. That is, the recognition epitope of 6B-10 antibody is different from that of 10C-3 antibody and G-81 antibody, and the site essential as the recognition epitope of 6B-10 antibody exists in the range of the amino acid sequence shown below. It was revealed.
Recognition epitope range: LEKGLDGAKKAVGGLG (66th to 81st amino acid sequence of full-length DCD)
In addition, the 6B-10 antibodies are Full-DCD, DCD-1L (SEQ ID NO: 2), SSL-46 (SEQ ID NO: 3), SSL-29 (SEQ ID NO: 4), SSL-25 (SEQ ID NO: 5), LEK- It was a monoclonal antibody that reacts with the peptide of 45 (SEQ ID NO: 6).
On the other hand, the 10C-3 antibody reacts with peptides of Full-DCD, DCD-1L (SEQ ID NO: 2), SSL-46 (SEQ ID NO: 3), LEK-45 (SEQ ID NO: 6), and SSL-29 (SEQ ID NO: 6). 4) It was a monoclonal antibody that did not react with the peptide of SSL-25 (SEQ ID NO: 5).
In addition, G-81 antibody is a peptide of Full-DCD, DCD-1L (SEQ ID NO: 2), SSL-46 (SEQ ID NO: 3), SSL-29 (SEQ ID NO: 4), LEK-45 (SEQ ID NO: 6). It was a monoclonal antibody that reacted and did not react with the peptide of SSL-25 (SEQ ID NO: 5).
Therefore, since the 6B-10 antibody, the 10C-3 antibody, and the G-81 antibody have different antigen recognizability, information about the distribution status of each cleavage type of DCD can be obtained by using them together. That is, information on individual differences in antibacterial activity can be obtained.
In addition, since the recognition epitopes of the 6B-10 antibody, the 10C-3 antibody, and the G-81 antibody are different, it may be applicable to a sandwich immunoassay method by using any two or more of them.

7). 2. Reactivity of anti-DCD monoclonal antibody (6B-10 antibody) to sweat DCD The reactivity of the 6B-10 antibody prepared in step 1 to DCD in a biological sample was confirmed by Western blot analysis using a sweat specimen.
The sweat specimen was obtained by collecting sweat generated on the face and back of a human (n = 6) due to exercise load. The sweat specimen was put into a Spectra / pore CE dialysis tube (MWCO: 2000, manufactured by Funakoshi), dialyzed in ultrapure water, and a part of the solution was concentrated 10 times using a centrifugal dryer. The concentrated and non-concentrated samples were developed on SDS-PAGE under reducing conditions, transferred to a PVDF membrane, and overnight at 4 ° C. with TBST containing 0.1% BSA (TBS containing 0.05% Tween 20, pH 7.4). After blocking, a 4000-fold diluted solution of 6B-10 antibody (3.3 mg / ml) as a primary antibody, a 4000-fold diluted solution of 10C-3 antibody (3.1 mg / ml), a 100-fold diluted solution of G-81 antibody, or Reaction was performed using a 2000-fold diluted solution of A-20 antibody, which is a commercially available goat polyclonal antibody, and alkaline phosphatase-labeled anti-mouse IgG antibody, mouse IgM antibody or anti-goat IgG antibody (all manufactured by CAPPEL) was used as a secondary antibody. Reacted.

After the PVDF membrane was washed with TBST, a mixed solution of NBT (nitroblue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3-indolyl phosphate) was added as a substrate for color development. As a result, as shown by the A-20 antibody, which is a polyclonal antibody, all specimens except for the specimen 5 have bands that can be detected as DCD. In 6B-10 antibody, a band of about 5 kDa was detected in Sample 1, Sample 2, Sample 4, and Sample 6 (FIG. 2). With the 10C-3 antibody, a band of about 5 kDa was detected in Sample 1, Sample 2, Sample 4, and Sample 6, and the same results as the 6B-10 antibody were shown.
From this result, it became clear that the 6B-10 antibody can recognize DCD, DCD complex or DCD binding protein that cannot be detected by G-81. In the sweat specimen used in this example, there was no difference in reactivity between the 6B-10 antibody and the 10C-3 antibody. In FIG. 2, lane M represents a molecular weight marker.

8). DCD quantification method using anti-DCD monoclonal antibody (6B-10 antibody) It was confirmed by enzyme immunoassay using synthetic peptide as an antigen that DCD could be quantitatively detected using 6B-10 antibody.
The enzyme immunoassay is the same as that described in 2. The antibody titer was measured by the same method.
First, a synthetic DCD-1L (manufactured by AnyGen) having a purity of 95% or more was diluted in two steps using a coating buffer (carbonated-bicarbonate buffer, pH 10) in a concentration range of 0.04 to 5.0 μg / ml, A 96-well EIA / RIA plate (Costar, High Binding) was dispensed (50 μL / well) and incubated at 4 ° C. overnight (about 14 hours) to physically adsorb synthetic DCD-1L to the plate.
The plate was washed 3 times with PBS containing 0.1% Tween 20 (hereinafter abbreviated as PBST), and then a blocking solution (PBS containing 0.5% BSA) was added at 100 μL / well and blocked at room temperature for 2 hours. After removing the blocking solution, an 8000-fold diluted solution (50 μl / well) of 6B-10 antibody (3.3 mg / ml) was dispensed as the primary antibody, and incubated at room temperature for 2 hours.
After the plate was washed 4 times with PBST, a peroxidase-labeled anti-mouse Ig (G + M) goat polyclonal antibody (manufactured by Tago Products) diluted as a secondary antibody was reacted at room temperature for 1 hour, and then orthophenylenediamine (manufactured by Sigma). A peroxidase substrate solution containing was added for color development. Ten minutes later, 1.5N sulfuric acid was added to stop color development, and then the absorbance at 492 nm was measured with a microplate reader.
As a result of plotting the obtained absorbance against the synthetic DCD-1L concentration, it was confirmed that linearity was obtained in the concentration range of 0.04 to 1.25 μg / ml (FIG. 3). Therefore, it was confirmed by the enzyme immunoassay method that the amount of DCD in the DCD-containing specimen can be quantified using the antibody of the present invention.

9. Quantification of DCD in sweat using anti-DCD monoclonal antibody (6B-10 antibody) (comparison with 10C-3 antibody)
It was confirmed by enzyme immunoassay that the DCD-degrading peptide contained in sweat can be detected with a larger detection amount than the 10C-3 antibody by using the 6B-10 antibody.
The enzyme immunoassay is the same as that described in 8. The same measurement method as described above was used.
The sweat specimen was obtained by collecting sweat generated on the human face and back due to exercise load on 40 adults. The sweat specimen was diluted 100-fold with Phosphate buffered saline (PBS (−), pH 7.0), and dispensed (50 μL / well) to a 96-well EIA / RIA plate (Costar, High Binding). Proteins and peptides contained in sweat were physically adsorbed to the plate by incubating at 0 ° C. overnight (about 14 hours).
The plate was washed 3 times with PBS containing 0.1% Tween 20 (hereinafter abbreviated as PBST), and then a blocking solution (PBS containing 0.5% BSA) was added at 100 μL / well and blocked at room temperature for 2 hours. After removing the blocking solution, dispense 50 μl / well of 8000-fold diluted solution of 6B-10 antibody (3.3 mg / ml) or 8000-fold diluted 10C-3 antibody (3.1 mg / ml) as the primary antibody, Incubated for 2 hours at room temperature.
After the plate was washed 4 times with PBST, a peroxidase-labeled anti-mouse Ig (G + M) goat polyclonal antibody (manufactured by Tago Products) diluted as a secondary antibody was reacted at room temperature for 1 hour, and then orthophenylenediamine (manufactured by Sigma). A peroxidase substrate solution containing was added for color development. Ten minutes later, 1.5N sulfuric acid was added to stop color development, and then the absorbance at 492 nm was measured with a microplate reader. A calibration curve was prepared using synthetic DCD-1L (manufactured by AnyGen) having a purity of 95% or more prepared to an appropriate concentration, and the concentration of DCD contained in sweat was calculated from the absorbance.
As a result, it was confirmed that in all 40 sweat specimens examined, the detected amount of DCD in sweat quantified with the 6B-10 antibody was larger than the detected amount with the 10C-3 antibody (FIG. 4). This Example showed that the detected amount of DCD-degrading peptide contained in sweat was larger in the 6B-10 antibody than in the 10C-3 antibody.
Since the 6B-10 antibody of the present invention recognizes the common sequence of all DCD-derived degraded peptides, the amount of total DCD peptide can be accurately quantified, and a degraded peptide that cannot be detected by the known 10C-3 antibody is detected. I was able to do that. In addition, the difference between the two bar graphs in FIG. 4 indicates that the amount of the degradable peptide that can be detected by the 6B-10 antibody is much larger than that of the 10C-3 antibody. If these two types of antibodies are used for detection, the total amount of DCD-degrading peptide in sweat is measured with 6B-10 antibody, and a certain group of DCD peptides (92 of full-length DCD) is detected with 10C-3 antibody. Since a peptide group having the amino acid sequence of the 108th to the 108th amino acid sequence (for example, SEQ ID NO: 2, 3, 6) is measured, It is also possible to calculate the ratio of 5).

  Since the monoclonal antibody (6B-10 antibody) of the present invention can specifically react with human DCD, it can be used for detection of human DCD. Moreover, the hybridoma of the present invention allows the antibody to be produced semi-permanently outside the cell and stably produced. The monoclonal antibody (6B-10 antibody) and the hybridoma producing the same of the present invention are useful for DCD analysis in human biological samples, prevention and treatment of infectious diseases derived from microorganisms, and their research.

FERM P- 22134

[Reference to deposited biological materials]
(1) FERM P- 22134 (6B-10 antibody-producing hybridoma)
The name and address of the depository that deposited the biological material AIST National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center 1-chome, East 1-chome, Tsukuba City, Ibaraki Prefecture, Japan 6
Date of deposit of biological material at the depository in Loi June 23, 2011 Deposit number FERMP P- 22134 attached by the depository in the high

Claims (5)

A monoclonal antibody produced by the hybridoma 6B-10 strain (FERM P-22134). 6B-10 strain (FERM P-22134) which is a hybridoma. A method for detecting DCD comprising the step of mixing a sample with the monoclonal antibody according to claim 1. The detection method according to claim 3, wherein the sample is derived from human sweat. A DCD detection kit comprising the monoclonal antibody according to claim 1 or the labeled monoclonal antibody.