ANTIBODY SPECIFIC TO PROFILAGGRIN C-TERMINAL DOMAIN, AND USE THEREOF

Abstract

The present invention provides an antibody specific to a C-terminal domain of a human profilaggrin gene, wherein the C-terminal domain is a peptide comprising the amino acid sequence set forth in SEQ ID NO:1 or a variant thereof, a method for detecting a filaggrin gene mutation using the antibody, and a kit for the detection.

Claims

1-5. (canceled)

6. A method of detecting a mutation in a filaggrin gene, comprising the steps of: obtaining a skin sample from a subject suffering from or suspected of having atopic dermatitis; determining whether a C-terminal domain of profilaggrin is present or absent in the sample, wherein the presence of the C-terminal domain of profilaggrin in the sample indicates that the filaggrin gene is not mutated and the absence of the C-terminal domain of profilaggrin in the sample indicates that the filaggrin gene is mutated, and subjecting the subject to a treatment or prevention of atopic dermatitis if being determined that the filaggrin gene is mutated.

7. The method of claim 6, wherein the C-terminal domain of profilaggrin comprises the amino acid sequence of SEQ ID NO: 1.

8. The method of claim 6, wherein the skin sample is obtained by tape stripping.

9. The method of claim 6, wherein the skin sample comprises a horny layer of the skin.

10. The method of claim 6, wherein determining whether the C-terminal domain of profilaggrin is present in the sample comprises performing an immunoassay.

11. The method of claim 10, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA).

12. The method of claim 10, wherein the immunoassay is a radioimmunoassay (RIA).

13. The method of claim 10, wherein the immunoassay is a Western blot.

14. The method of claim 6, wherein determining whether the C-terminal domain of profilaggrin is present in the sample comprises immunostaining.

14. The method of claim 6, wherein the mutation in the filaggrin gene results in termination of a filaggrin protein at amino acid positions 2889 or 4022.

16. The method of claim 6, wherein an absence of the C-terminal domain of profilaggrin in the sample indicates the filaggrin gene is mutated.

17. The method of claim 6, wherein the subject is a human.

18. A method of detecting a mutation in a filaggrin gene, comprising the steps of (a) obtaining a skin sample comprising a horny layer of the skin from a human subject, (b) detecting a C-terminal domain of profilaggrin in the sample by contacting the sample with an antibody that binds to the C-terminal domain of profilaggrin; wherein an inability to detect the C-terminal domain of profilaggrin in the sample indicates the filaggrin gene is mutated, and (c) subjecting the subject to a treatment or prevention of atopic dermatitis if being determined that the filaggrin gene is mutated.

19. The method of claim 18, wherein the human subject is suffering from or suspected of having atopic dermatitis.

20. The method of claim 18, wherein the C-terminal domain of profilaggrin comprises the amino acid sequence of SEQ ID NO: 1.

21. The method of claim 18, wherein the skin sample is obtained by tape stripping.

22. The method of claim 18, wherein contacting the sample with the antibody that binds to the C-terminal domain of profilaggrin is performed as part of an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), a Western blot, or an immunostain.

23. The method of claim 18, wherein the mutation in the filaggrin gene results in termination of a filaggrin protein at amino acid positions 2889 or 4022.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] [FIG. 1] is a schematic illustration showing a structure of the profilaggrin gene (Chrlq21.3/Human) having 12 filaggrin repeats, and mutation sites of the gene.

[0025] [FIG. 2] is an immunostaining diagram of normal skin (normal (+/+)) without any profilaggrin gene mutations and of atopic dermatitis skin (AD(+/+)). The drawings on the left side show the results of staining with Hematoxylin-Eosin (HE staining), and the drawings on the right side show the results of staining with an antibody specific to the C-terminal domain of profilaggrin and a chromogenic substrate DAB (3,3-diaminobenzidine) (FLG-C).

[0026] [FIG. 3] is an immunostaining diagram of atopic dermatitis skin with profilaggrin gene mutations (S2889X; K4022X). The drawings on the left side show the results of staining with Hematoxylin-Eosin (HE staining), and the drawings on the right side show the results of staining with the antibody specific to the C-terminal domain of profilaggrin and a chromogenic substrate DAB (3,3-diaminobenzidine) (FLG-C).

EMBODIMENT FOR CARRYING OUT THE INVENTION

[0027] According to a first aspect of the present invention, there is provided an antibody specific to a C-terminal domain of a human profilaggrin gene, wherein the C-terminal domain is a peptide comprising the amino acid sequence set forth in SEQ ID NO:1 or a variant thereof. Filaggrin is roughly categorized into Type I and Type II. However, the term profilaggrin as used herein refers to Type I. Profilaggrin has a structure comprising filaggrin repeats consisting of 10 to 12 continuous filaggrin molecules interposed between the N-terminal domain and the C-terminal domain (FIG. 1). SEQ ID NO:2 indicates the entire sequence of a C-terminal domain consisting of 157 amino acids.

[0028] With respect to the C-terminal domain, the amino acid sequence set forth in SEQ ID NO:1 is not homologous to either Type II profilaggrin or hornerin, which is a profilaggrin-analogous molecule, and is a sequence specific to Type I. Therefore, an antibody specific to the amino acid sequence does not cross-react with these analogous proteins.

[0029] The variant may be any variant as long as an antibody obtained using the variant as an antigen does not recognize a peptide having a sequence analogous to the amino acid sequence set forth in SEQ ID NO:1 in the skin and is specific to the C-terminal domain of a human profilaggrin gene. For example, the variant may be a peptide comprising an amino acid sequence derived from the amino acid sequence set forth in SEQ ID NO:1 by deletion, substitution, or addition of one or more amino acids.

[0030] While not limited thereto, the peptide can be obtained by chemically synthesis according to a conventional method. The antibody of the present invention can be obtained by administering the peptide as the antigen, preferably an antigen complex comprising the peptide bound to a suitable carrier, to mammals, for example, rats, mice, rabbits, etc. While not limited thereto, the dosage amount of the antigen or antigen complex per animal is 0.1 to 100 mg when no adjuvant is used, and is 10 to 1000 g when an adjuvant is used. Examples of the adjuvant include Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvant (FIA), aluminum hydroxide adjuvant, etc.

[0031] The immunization is mainly performed by intravenous, subcutaneous, or intraperitoneal injection. Further, the immunization interval is not specifically limited. However, immunization is performed 1 to 10 times, preferably 2 to 5 times, at an interval of several days to several weeks, preferably, an interval of 2 to 5 weeks. Moreover, the antibody titer is measured by Western blotting, Enzyme-Linked Immunosorbent Assay (ELISA or EIA), Radioimmunoassay (RIA), etc., 6 to 60 days after the day of the final immunization. Preferably, blood is collected and antiserum is obtained on the day when the maximum antibody titer is exhibited.

[0032] The polyclonal antibody may be purified by affinity chromatography using a column to which the antigen peptides are bound, and other purification methods known to a person skilled in the art, for example, ion exchange chromatography, gel electrophoresis chromatography, high-performance liquid chromatography, etc. Further, the antibody of the present invention may be a monoclonal antibody. The monoclonal antibody may be prepared according to an already known method using a peptide having the amino acid sequence set forth in SEQ ID NO:1 or a variant thereof.

[0033] According to a second aspect of the present invention, there is provided a method for detecting a mutation in a filaggrin gene, wherein the method comprises the steps of: detecting the presence or absence of said C-terminal domain in a skin sample of a subject with the antibody, and judging that the mutation in a filaggrin gene is present in the subject, in case when said C-terminal domain is not detected. The term a mutation in a filaggrin gene as used herein refers to one or more mutations of a filaggrin gene, which inhibit the formation of the C-terminal domain of profilaggrin. Examples of the mutations include the mutation S2889X in which termination occurs at the serine in position 2889, the mutation K4022X in which termination occurs at the lysine in position 4022, etc. (FIG. 1).

[0034] A mutation in the profilaggrin gene leads to atopic dermatitis. Therefore, according to the method of the present invention, when the C-terminal domain is not detected, it is judged that the subject is suffering or is highly likely to suffer from atopic dermatitis.

[0035] The detection of the presence or absence of the C-terminal domain is carried out by an immunological method well known to a person skilled in the art, for example, immunoassay (ELISA, RIA, etc.) The skin samples are collected by any method from the arms, legs, head, etc., of subjects. The subjects are mammals, preferably humans with suspected skin diseases caused by filaggrin gene mutations, specifically, atopic dermatitis. The method for collecting the skin samples is not specifically limited, but a tape stripping method is preferable because it is simple and non-invasive. The tape stripping is a method for collecting a horny layer sample, which comprises bonding a piece of adhesive tape to the skin surface layer and peeling off the piece so that the skin horny layer adheres to the peeled adhesive tape. According to the tape stripping method, a skin sample containing a profilaggrin gene can be simply obtained, and a filaggrin gene mutation can be detected non-invasively.

[0036] A preferable method of tape stripping is as follows: First, a surface layer of skin is cleaned with, for example, ethanol, etc., to remove sebum, dirt, and the like. A piece of adhesive tape cut to a suitable size (for example 55 cm) is gently placed on the skin surface. The entire piece of tape is pressed flat onto the skin surface by applying uniform pressure, and the adhesive tape is subsequently peeled off while applying uniform force. The adhesive tape may be a commercially available cellophane tape, for example, Scotch Superstrength Mailing Tape (manufactured by 3M), Cellophane Tape (CelloTape(R), Nichiban), etc.

[0037] A neutral to weakly alkaline buffer including a non-ionic surfactant can be used to extract a soluble component from the tape-stripped horny layer, and includes, but not limited to the following buffer: 100 mM Tris HCl+0.14 M NaCl+0.1% Triton 100]. The tape having a horny layer adhering thereto is cut into a strip with scissors, etc., transferred to a container, such as an Eppendorf tube, immersed in a small amount of the aforementioned buffer, stirred by inversion, to efficiently extract the soluble components. The extracted soluble components are subjected to an immunoassay, etc., using the antibody of the present invention, to judge the presence or absence of a profilaggrin gene mutation.

[0038] According to a third aspect of the present invention, there is provided a kit for detecting a mutation in a filaggrin gene, comprising the antibody. The kit may comprise the antibody of the present invention in a container. The kit may further comprise reagents required for the aforementioned detection method, for example, in case of the detection of a mutation by the ELISA method, an enzyme conjugate, substrate solution, quenching solution, wash solution, enzyme conjugate diluent, assay buffer, control, sample diluent, conjugate, etc., and operating instructions explaining the procedures of the method.

[0039] The present invention will be specifically explained below in more detail with reference to specific examples. However, the present invention is not limited thereto.

EXAMPLES

[0040] Immunohistochemical Staining

[0041] Skin section samples were prepared from normal skin and atopic dermatitis skin, which were free of a profilaggrin gene mutation, and atopic dermatitis skin with a profilaggrin gene mutation (S2889X or K422X). The immunohistochemical staining of the skin fragment was performed by the method described in Kamata et al. (J. Biol. Chem., Vol. 284, Issue 19, 12829-12836, May 8, 2009). The antibody specific to the profilaggrin C-terminal domain was obtained by immunizing a rabbit with a synthetic peptide having the amino acid sequence CKASAFGKDHPRYYATYINKDP (SEQ ID NO: 1) as the immunogen (manufactured by Sigma Inc.)

[0042] The normal skin and atopic dermatitis skin, which were free of a profilaggrin gene mutation, were stained with Hematoxylin-Eosin, or with the antibody specific to a profilaggrin C-terminal domain and a chromogenic substrate DAB (3,3-diaminobenzidine). The results are shown in FIG. 2. Profilaggrin was localized in the granular layer. The color development of DAB staining in the granular layer was reduced in the atopic dermatitis skin free of a profilaggrin gene mutation compared to normal skin (refer to the immunostaining diagram of FLG-C). Namely, the results of FIG. 2 reveal that the expression of filaggrin is reduced in atopic dermatitis skin even free of a profilaggrin gene mutation.

[0043] Next, immunostaining was performed for atopic dermatitis skin with the profilaggrin gene mutation S2889X or K422X. In the same manner as the results in FIG. 2, each skin was stained with Hematoxylin-Eosin (left side: HE stain), or with the antibody specific to a profilaggrin C-terminal domain and a chromogenic substrate DAB (3,3-diaminobenzidine) (right side: FLG-C), and the results are shown in FIG. 3. The granular layer was not stained with DAB unlike the results in FIG. 2. Namely, it was revealed that the profilaggrin C-terminal was not formed by the mutation in the atopic dermatitis skin with the mutation.

[0044] The aforementioned results have revealed that the antibody specific to the profilaggrin gene C-terminal domain of the present invention could be used to simply detect the presence or absence of filaggrin gene mutations known to be many, without verifying every mutation by sequencing, and thereby to judge that the skin of a subject is suffering or is highly likely to suffer from atopic dermatitis.