JP5565723B2 - Anti-DCD monoclonal antibody - Google Patents

Description

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

  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, Enterococcus, Staphylococcus aureus and Candida under the same pH and the same salt concentration as human sweat, and it is secreted constantly in 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 monoclonal antibody, there is an IgM class anti-DCD monoclonal antibody (G-81) produced by Sagawa et al. Of Wakayama Medical University (Non-patent Document 4), but an IgG class antibody has not yet been provided. The reason for this is not clear, but the antigenicity of DCD itself is very weak, and it is thought that IgG antibody-producing cells cannot be obtained by simple immunization and hybridoma screening techniques. However, the IgG class antibody is generally more specific, and the DCD is more accurate because the antigen aggregation and complement activation are stronger in the IgM class antibody. For detection, IgG class antibodies are preferred. In particular, the IgG class antibody is suitable for the above-described method for analyzing a large number of parameters using a plurality of antibodies and beads, and the performance and advantages of this technique are utilized with an IgM class antibody. There is a problem that cannot be done at all.

U.S. Pat. No. 7,348,409

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.

  It is an object of the present invention to provide a hybridoma that produces an IgG class monoclonal antibody that recognizes human DCD and 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 have produced human DCD recombinantly in Escherichia coli and produced a large amount thereof, and antibody-producing cells obtained from animals immunized as antigens thereof. And hybridomas capable of producing IgG class monoclonal antibodies recognizing human DCD were established by fusing myeloma cells by the cell fusion method and screening for hybridomas having the desired reactivity and producing antibodies. . 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 monoclonal antibody that recognizes human DCD.
(2) The monoclonal antibody according to (1) above, which is obtained using a His-tag fusion human DCD as an antigen.
(3) An IgG class monoclonal antibody that recognizes human DCD and reacts with the peptide sequences of SEQ ID NOs: 2, 3, and 6 but does not react with the peptide sequences of SEQ ID NOs: 4 and 5.
(4) The monoclonal antibody according to (1), which is produced by the 10C-3 strain (FERM AP-21792).
(5) A hybridoma capable of producing an IgG class monoclonal antibody that recognizes human DCD.
(6) 10C-3 strain (FERM AP-21792), which is the hybridoma according to (5) above.
(7) A method for producing an IgG class monoclonal antibody that recognizes human DCD, wherein the hybridoma according to (5) or (6) above is used.
(8) The hybridoma according to the above (5) or (6) is grown in a liquid medium or in the peritoneal cavity of a constant temperature animal (animal species in which the hybridoma is not immunologically excluded) excluding humans, and a monoclonal antibody is produced and accumulated. A method for producing an IgG class monoclonal antibody that recognizes human DCD, comprising collecting and collecting the same.
(9) A method for examining human DCD, wherein the monoclonal antibody according to (1) is used.
(10) A human DCD test kit using the monoclonal antibody according to any one of (1) to (4) or a labeled monoclonal antibody.

  According to the present invention, it has become possible to provide an IgG class monoclonal antibody capable of detecting human 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 (10C-3 antibody) (Example). It is the figure which confirmed and showed the reactivity with anti- DCD monoclonal antibody (10C-3 antibody) to DCD in sweat (Example). It is the figure which showed the analytical curve in the determination method of DCD using an anti- DCD monoclonal antibody (10C-3 antibody) (Example).

The “monoclonal antibody” of the present invention may be any monoclonal antibody as long as it is an IgG class monoclonal antibody that recognizes human DCD. In addition to the whole antibody molecule, it may be a fragment of an antibody that can recognize human DCD.
Here, “recognizing human DCD” means binding to human DCD using human DCD as an antigen, or performing some action by binding.
The human DCD recognized as an antigen may be the entire human DCD or a part thereof.
Such a “monoclonal antibody” includes a monoclonal antibody that recognizes human DCD that reacts with the peptide sequences of SEQ ID NO: 2, 3, 6 and does not react with the peptide sequences of SEQ ID NO: 4, 5; -3 strain (FERM AP-21792). Since the monoclonal antibody produced by the 10C-3 strain (FERM AP-21792) is an IgG class monoclonal antibody, its specificity is high and preferable compared to an IgM class antibody. 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 may be any hybridoma as long as it is capable of producing an IgG class monoclonal antibody that recognizes human DCD. Examples of such hybridoma include 10C-3 strain (FERM AP-21792).
The “hybridoma” of the present invention uses human DCD as an immunizing antigen to fuse spleen cells excised from the spleen of mice, rats, etc., whose antibody titers in serum are sufficiently increased, and myeloma cells, etc. Is preferably obtained by culturing 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 immune antigens for obtaining hybridomas include human DCD and His-tag fusion human DCD.
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 “method for producing a monoclonal antibody” 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 human DCD.
For example, a hybridoma capable of producing an IgG class monoclonal antibody recognizing human DCD is grown in a liquid medium or in the abdominal cavity of a constant temperature animal excluding humans, and a monoclonal antibody is produced, accumulated, and collected. Can be mentioned. Examples of the “constant animals other than humans” include animal species that do not immunologically exclude hybridomas in non-human constant temperature animals, such as mice that are syngeneic with myeloma cells, nude mice, nude rats, and the like.

The “human DCD test method” of the present invention may be any method as long as it uses a IgG class monoclonal antibody that recognizes human DCD for the test.
For example, in the test method of the present invention, by performing an enzyme immunoassay using an IgG class monoclonal antibody that recognizes human DCD, human DCD contained in a sample such as sweat can be detected and quantified.
In addition, a method of combining a IgG antibody that recognizes human DCD of the present invention with another antibody and testing a number of parameters with high sensitivity at once using a plurality of beads to which these antibodies are bound ( 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 degraded DCD can also be examined.

The “test kit” of the present invention refers to a kit for testing human DCD, and any kit that uses an IgG class monoclonal antibody that recognizes human DCD may be used. For example, a kit using a combination of an IgG class monoclonal antibody that recognizes human 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 the detection and quantification of human 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 Production of Antigen Protein As an immunizing antigen, a fusion protein of approximately 16 kDa polyhistidine (hereinafter abbreviated as His-tag) and DCD recombinantly expressed in E. coli 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).
In general, in order to increase antigenicity, a technique of adding a carrier protein such as bovine serum albumin (BSA) or keyhole limpet hemocyanin to the antigen protein is used. In the present invention, an antibody against the carrier protein is used. In order to prevent the production of bismuth, the His-tag fusion DCD was used as an antigen as it was without adding a carrier protein.

2. Production of hybridoma PBS solution of antigen protein (antigen protein concentration: 3.5 mg / ml) prepared in step 1 was mixed with adjuvant TiterMax Gold (manufactured by CytRx) in an equal amount, and the amount of antigen was 150 μg / mouse in W / O micelle state. Mice (BALB / C, 7 weeks old) were administered subcutaneously on the back. Further, similarly, an antigen mixed with TiterMax Gold was boosted twice at intervals of 10 to 14 days. Two weeks after the last boost, the antigen protein was mixed in an equal volume 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.

  The antibody titer was confirmed by enzyme immunoassay using His-tag fusion DCD as a solid phase. First, using a coating buffer (carbonate-bicarbonate buffer, pH 10), the His-tag fusion DCD was diluted to 10 μg / ml, and dispensed to a 96-well EIA / RIA plate (Costar, High Binding) (50 μL / Well) and physical incubation 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 sera from immunized mice was 1.8, 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.

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 (molecular weight 1400 to 1600, manufactured by Sigma) ) The cells were fused in the presence.
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 about 2 weeks later, the primary antibody was cultured in the enzyme immunoassay shown in the above antibody titer measurement for the culture supernatant of the well in which the hybridoma had grown. From the experiment in which the supernatant was replaced, a strain that was promising to produce an antibody that reacted with the His-tag fusion DCD and did not react with the synthetic polyhistidine (6 ×) 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 11 types of anti-DCD monoclonal antibody-producing hybridomas were produced. Established. Of the 11 species, 10 were of the IgM class and 1 was of the IgG class. This IgG-class hybridoma was deposited on March 24, 2009 by the applicant at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1st, 1st, 1st East, Tsukuba City, Ibaraki, Japan). , A receipt number (FERM AP-21792) is assigned. Hereinafter, the anti-DCD monoclonal antibody produced by this hybridoma (FERM AP-21792) is referred to as 10C-3 antibody.

3. Production of Monoclonal Antibody A 12-week-old female BALB / C mouse that had been injected intraperitoneally with 0.5 mL of pristane 2 weeks before the administration of the hybridoma was treated with 5 × 10 ⁶ cells of the hybridoma obtained above. The dose was administered intraperitoneally. About 10 days later, 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 (10C-3 antibody) was purified.

4). Identification of immunoglobulin class and subclass of anti-DCD monoclonal antibody (10C-3 antibody) The immunoglobulin class of the 10C-3 antibody prepared in Step 1 was confirmed using the Mouse Monoclonal Antibody Isotyping Test Kit (manufactured by Serotec).
The 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 put into a tube, and the immunoglobulin class was determined by the position of the band appearing on the strip after 10 minutes. As a result, it was confirmed that the immunoglobulin class of the 10C-3 antibody is the IgG2b, κ light chain desired in the present invention.

  Hereinafter, the 10C-3 antibody obtained by the present invention and the conventional IgM class anti-DCD mouse monoclonal antibody (hybridoma culture supernatant provided by Wakayama Medical University, hereinafter referred to as G-81 antibody) And the antigen recognition specificity of the 10C-3 antibody was confirmed.

5. 2. Confirmation of antigen recognition specificity of anti-DCD monoclonal antibody (10C-3 antibody) Western blot analysis was performed to confirm the specificity of antigen recognition of the 10C-3 antibody prepared in (1). Above 1. The His-tag fusion DCD prepared in step 1 was developed on SDS-PAGE under reducing conditions, transferred to a PVDF membrane, and TBST containing 0.1% BSA (TBS containing 0.05% Tween 20, pH 7.4) at 4 ° C. After blocking for 1 day, the 8000-fold diluted solution of 10C-3 antibody (3.3 mg / ml) or the 10-fold diluted solution of G-81 antibody as the primary antibody, alkaline phosphatase-labeled anti-mouse IgG antibody (CAPPEL) as the secondary antibody Made). 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 10C-3 antibody reacted with the His-tag fusion DCD in the same manner as the G-81 antibody (FIG. 1). In FIG. 1, lane M represents the molecular weight marker, lane A represents the Coomassie blue (CBB) staining result of His-tag fusion DCD on the PVDF membrane, lane B represents the 10C-3 antibody, and lane C represents G-81. The result of the Western blot analysis by an antibody is represented.

6). Identification of recognition epitope of anti-DCD monoclonal antibody (10C-3 antibody) In order to confirm the recognition epitope of the 10C-3 antibody prepared in step 1, the amino acid sequences shown in Table 1 from peptides (non-patent document 3) that are known to exist in sweat as a degraded form of DCD A synthetic peptide was prepared by selection (manufactured by AnyGen), and the reactivity of the 10C-3 antibody was confirmed by enzyme immunoassay using these as antigens. These sequences are shown in SEQ ID NOs: 2 to 6 in Sequence Listing.

The enzyme immunoassay was performed by the following method.
First, each synthetic peptide was diluted to 100 pmol / ml ) using -PBS (−), and dispensed (50 μL / well) into a 96-well EIA / RIA plate (Costar, High Binding) for about 18 hours at room temperature. Physical adsorption was achieved by incubation.
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, the hybridoma culture supernatant of 10C-3 antibody or G-81 antibody (50 μl / well) was dispensed as the primary antibody 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 10C-3 antibody and the G-81 antibody to the synthetic peptide was as shown in Table 2, and the recognition epitope of the 10C-3 antibody was different from that of the G-81 antibody. It has been clarified that the site essential as a recognition epitope exists within the range of the amino acid sequence shown below.
Recognition epitope range: ESVGKGAVHDVKDVLDS (92nd to 108th amino acid sequence of full length DCD)
The 10C-3 antibody reacts with the peptides Full-DCD, DCD-1L (SEQ ID NO: 2), SSL-46 (SEQ ID NO: 3), LEK-45 (SEQ ID NO: 6), SSL-29 (SEQ ID NO: 4), It was a monoclonal antibody that did not react with the peptide of SSL-25 (SEQ ID NO: 5).
On the other hand, 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 10C-3 antibody and the G-81 antibody have different antigen recognition properties, information about the structure of the DCD fragment can be obtained by using them together.
In addition, since the recognition epitopes of the 10C-3 antibody and the G-81 antibody are different, it may be applicable to a sandwich immunoassay method.

7). 2. Reactivity of anti-DCD monoclonal antibody (10C-3 antibody) to sweat DCD The reactivity of the 10C-3 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 = 3) 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, the reaction is carried out using a 8000-fold diluted solution of 10C-3 antibody (3.3 mg / ml) or a 10-fold diluted solution of G-81 antibody as a primary antibody, and an alkaline phosphatase-labeled anti-mouse IgG antibody or a secondary antibody. Mouse IgM antibodies (both manufactured by CAPPEL) were reacted with each other.

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, the 10C-3 antibody detected more bands in the sweat specimen than the G-81 antibody (FIG. 2).
In particular, the approximately 18 kDa band of Specimen 1, Specimen 2 and Specimen 3 and the approximately 32 kDa band of Specimen 2 were not detected by G-81 antibody, but were detected by 10C-3 antibody. From this result, it became clear that the 10C-3 antibody can recognize DCD, DCD complex or DCD binding protein that cannot be detected by G-81. In FIG. 2, lane M represents a molecular weight marker, and lane PC represents a His-tag fusion DCD used as a positive control in this analysis.

8). DCD Quantification Method Using Anti-DCD Monoclonal Antibody (10C-3 Antibody) It was confirmed by enzymatic immunoassay using a synthetic peptide as an antigen that DCD could be quantitatively detected using 10C-3 antibody.
The enzyme immunoassay is the same as that described in 2. It confirmed by the method similar to the measurement of the antibody titer.
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 physically adsorbed 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 removing the blocking solution, an 8000-fold diluted solution (50 μl / well) of 10C-3 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 amount of protein, it was confirmed that linearity was obtained in a concentration range of 0.04 to 2.5 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.

  Since the human monoclonal antibody (10C-3 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 human monoclonal antibody (10C-3 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.

Claims (3)

10C-3 strain (FERM P-21792) is produced eggplant makes the chromophore at the distal end monoclonal antibodies. A method for detecting human dermicidin in a sample, wherein the monoclonal antibody according to claim 1 is used. A human dermisidine detection kit using the monoclonal antibody according to claim 1 or the labeled monoclonal antibody.