JP6586450B2 - Allergen antibody and composition having the same - Google Patents

Description

  The present invention relates to an antibody obtained from house dust as an antigen, a composition thereof, and a cosmetic having the antibody.

There are various allergens in human living space. These include pollen allergens, dog and cat allergens, mite allergens and house dust allergens. These are said to be involved in allergic diseases and skin diseases. Allergens in dogs, cats, ticks and house dust are thought to induce asthma and atopic dermatitis.

  Patent Document 1 discloses an antibody against cypress pollen allergen.

International Publication No. 2012/105541

As in Patent Document 1, in the case where there is one antigen, there are those that describe antibodies against this.
However, in the case of allergies occurring in the living room, there are cases where the antigens are spread over a plurality of mite allergens, dogs and cat allergens. It takes time to produce antibodies corresponding to all of these. There was no report on the effectiveness of 1 antibody.

In addition, there has been no report of an antibody capable of alleviating the disease against bacteria related to the diseases occurring in the skin.

  The present invention has been conceived in view of the above problems, and is expected to reduce skin moisturization caused by Pseudomonas aeruginosa among diseases occurring in the skin and relieve skin aging and atopic dermatitis. It is an antibody.

  The antibody according to the present invention is obtained by inoculating an ostrich female with a plurality of allergens as antigens, and obtained from an egg obtained from the female ostrich, and has an adsorption reaction to almost all of the inoculated antigens. Therefore, allergens in living space can be inactivated with one antibody.

By including these antibodies in sprays, cosmetics (skin care products), sprays, and pharmaceuticals, allergens can be reduced by causing antigen-antibody reactions with allergens and neutralizing them. Mitigation can be achieved. Further, these antibodies may be carried on an air conditioner filter or a mask filter. Further, sprays and sprays may be sprayed not only by a person but also from a machine such as an air conditioner.

Hereinafter, the antibody according to the present invention will be described. The following description shows one embodiment of the present invention, and the following embodiment and examples may be modified without departing from the spirit of the present invention.

A plurality of types of antigens that can be used in the present invention can be mixed, and various types of mite allergens, specific allergens such as dog allergens and cat allergens, and house dust allergens may be used as a total thereof. A house dust allergen is an aggregate considered as an allergen collected from within the living space. Therefore, the house dust allergen may contain mite allergens, dog allergens, and cat allergens. The house dust allergen is an allergen related to house dust, and may be referred to as an aggregate of a plurality of allergens.

Moreover, a Pseudomonas aeruginosa homogenate liquid can also be used for an antigen. Pseudomonas aeruginosa is a bacterium that produces ceramidase. This ceramidase is an enzyme that degrades ceramide, which is a molecule for maintaining the barrier function of the skin. Therefore, when ceramide is decomposed and deficient, the barrier function of the skin is lowered, the moisture retention of the skin is lowered, and it causes aging of the skin and atopic dermatitis.

Ceramide deficiency is caused by reduced production of ceramide and accelerated degradation of ceramide. In addition, it is considered that ceramide is excessively decomposed by ceramidase from skin bacteria such as Pseudomonas aeruginosa, leading to reduced skin moisture retention, aging, and induction of atopy.

The antibody against Pseudomonas aeruginosa homogenate binds not only to Pseudomonas aeruginosa but also to ceramidase produced by Pseudomonas aeruginosa as shown in the examples described later. Therefore, the growth of Pseudomonas aeruginosa is suppressed, the activity of ceramidase is also inhibited, and the degradation of ceramide is suppressed. Therefore, ceramide is maintained, and as a result, the skin environment is considered to be improved.

The present invention uses avian females. If it is birds, there will be no restriction | limiting in particular. However, ostriches are suitable for mass production because many antibodies can be obtained in one lot.

The antibody which concerns on this invention can reduce the effect as an allergen by providing the antibody itself to an allergen. The method of application may be included in a liquid and sprayed in the form of a mist, or may be sprayed together with an aerosol (gas dispersion system). Therefore, you may include in a deodorizer and an aromatic. When Pseudomonas aeruginosa is used as an antigen, the antibody can be directly applied to the skin. It may also be included in lotions and cosmetics. Moreover, you may make it catch the allergen in inhalation by including in the filter of an air conditioner or a mask.

Therefore, the antibody according to the present invention may be used as one component of the composition. Examples of the composition include skin moisturizing cosmetics, anti-aging cosmetics, atopy cosmetics, and pharmaceuticals corresponding thereto.

<Reactivity to mite allergens of egg yolk antibody prepared using ostrich, chicken, quail>
An antibody against mite allergen was prepared as follows. Mature female bird (ostrich,
Chicken, quail). Mite allergen (Dfb, Dpb, Dff, Dpf) 50
μg was mixed with 0.2 mL of Freund's complete adjuvant and the ostrich was first immunized. Each antigen was individually inoculated into 5 ostriches, 5 chickens and 5 quails. Ostriches, chickens and quails have been vaccinated with the same amount of antigen.

After the first immunization, each bird was boosted with a mixture of 50 μg of antigen and Freund's incomplete adjuvant at 2 and 4 weeks. The egg yolk antibody (IgY) was purified from the egg yolk of each bird obtained 8 weeks after the first immunization. The reactivity of the obtained egg yolk antibody was determined by ELISA (Enzyme-L
verified by Inked Immuno Sorbent Assay).

The reactivity of antibodies to allergens by ELISA was examined as follows. 10 μg of each mite allergen was separately solidified in each hole of a 96-well ELISA plate (4 hours at room temperature). Then ostrich antibody (mixture of antibodies from egg yolk obtained from each of three ostriches), chicken antibody (mixture of antibodies from egg yolk obtained from each of three chickens), quail antibody (obtained from three quails each) A mixture of antibodies from the egg yolks) was added dropwise to each well and reacted at room temperature for 1 hour. After washing, the HRP-labeled secondary antibody for each antibody was reacted at room temperature for 1 hour. I let you.

After sufficient washing, absorbance (450 nm) was measured with a plate reader using a peroxidase coloring kit (S-Bio SUMILON). The maximum dilution factor showing the absorbance value of 2 times or more of the yolk antibody of each bird species before immunization was shown as the ELISA value. The results are shown in Table 1.

It became clear that highly sensitive egg yolk antibodies were produced by immunizing ostriches, chickens, and quails with mite allergens (Dfb, Dpb, Dff, Dpf). In particular, despite the immunization of each bird species with the same amount of antigen, giant ostriches produced the most reactive antibodies. In other words, ostriches can produce highly sensitive antibodies with a small amount of antigen.

<Reactivity of egg yolk antibody prepared by immunizing ostrich, chicken and quail with house dust allergen>
An antibody against house dust was prepared as follows. Mature female bird (ostrich,
Chicken, quail). The ostrich was first immunized by mixing 50 μg of house dust allergen with 0.2 mL of Freund's complete adjuvant. Each antigen was individually inoculated into 5 ostriches, 5 chickens and 5 quails. Ostriches, chickens and quails have been vaccinated with the same amount of antigen. Here, the house dust allergen is a collection of substances considered to be allergens collected from living space.

After the first immunization, each bird was boosted with a mixture of 50 μg of antigen and Freund's incomplete adjuvant at 2 and 4 weeks. The egg yolk antibody (IgY) was purified from the egg yolk of each bird obtained 8 weeks after the first immunization. Mite allergen and cat allergen of the obtained egg yolk antibody,
Reactivity to canine allergens was determined by ELISA (Enzyme-Linked Immun.
o Sorbent Assay).

The reactivity of the antibody to house dust allergen by ELISA was examined as follows. Each mite allergen (Dfb, Dpb, Dff,
Dpf) and cat allergen nFel1, and canine allergen nCanf1 were each separately solidified (4 hours at room temperature). Then ostrich antibody (mixture of antibodies from egg yolk obtained from each of three ostriches), chicken antibody (mixture of antibodies from egg yolk obtained from each of three chickens), quail antibody (obtained from three quails each) Mixture of antibodies from egg yolk)
(2 mg / mL stock solution) was dropped into each hole and allowed to react at room temperature for 1 hour.

After washing, an HRP-labeled secondary antibody for each antibody was reacted at room temperature for 1 hour. After sufficient washing, absorbance (450 nm) was measured with a plate reader using a peroxidase coloring kit (S-Bio SUMILON). The maximum dilution factor showing the absorbance value of 2 times or more of the yolk antibody of each bird species before immunization was shown as the ELISA value. The results are shown in Table 2.

By immunizing house dust allergens, it was found that highly sensitive egg yolk antibodies against mite allergens (Dfb, Dpb, Dff, Dpf) and cat allergen nFel1 and canine allergen nCanf1 were produced in ostriches, chickens and quails. In particular, despite the immunization of each bird species with the same amount of antigen, giant ostriches produced the most reactive antibodies. This shows that if ostriches are used, highly sensitive antibodies can be produced with a small amount of antigen.

<Allergic reaction suppression effect of egg yolk antibody prepared by immunizing ostrich with house dust allergen>
The effect of egg yolk antibody obtained using house dust allergen as an antigen was examined. Disc filter paper soaked with house dust allergen (contains 5 μg of allergen in one disc)
(Product name: Allergen disc “Torii” house dust) 5 μL PBS (phosphate buffer) or 5 μL ostrich egg yolk antibody (produced by immunizing mite allergen Dfb or house dust allergen) (15 mg / mL) And left at room temperature for 10 minutes. After that, put the disc filter paper on the lower turbinate mucosa of volunteers with house dust allergy, leave it for 5 minutes, observe the presence or absence of clinical symptoms (nasal itching, sneezing, aqueous rhinorrhea, mucosal swelling) Another expression rate was calculated. The results are shown in Table 3.

The ostrich egg yolk antibody prepared by immunizing mite allergen and house dust allergen was found to reduce the allergic reaction.

<Reactivity of egg yolk antibody prepared by immunizing ostrich, chicken, quail with Pseudomonas aeruginosa homogenate>
An antibody using a Pseudomonas aeruginosa homogenate solution as an antigen was prepared as follows. Mature female birds (ostrich, chicken, quail) were used. Pseudomonas aeruginosa homogenate solution (protein amount 50 μg) was mixed with Freund's complete adjuvant 0.2 mL, and the ostrich was first immunized. Each antigen was individually inoculated into 5 ostriches, 5 chickens and 5 quails. Ostriches, chickens and quails have been vaccinated with the same amount of antigen.

After the first immunization, each bird was boosted with a mixture of 50 μg of antigen and Freund's incomplete adjuvant at 2 and 4 weeks. The egg yolk antibody (IgY) was purified from the egg yolk of each bird obtained 8 weeks after the first immunization. The reactivity of the obtained egg yolk antibody to Pseudomonas aeruginosa (bacteria) and ceramidase was verified by ELISA.

The reactivity of the antibody to Pseudomonas aeruginosa homogenate by ELISA was examined as follows. 10 Pseudomonas aeruginosa (bacteria) and ceramidase in each well of the 96-well ELISA plate
μg was layered separately (4 hours at room temperature). Then ostrich antibody (mixture of antibodies from egg yolk obtained from each of three ostriches), chicken antibody (mixture of antibodies from egg yolk obtained from each of three chickens), quail antibody (obtained from three quails each) A mixture of antibodies from egg yolks) was added dropwise to each well and allowed to react at room temperature for 1 hour.

After washing, an HRP-labeled secondary antibody for each antibody was reacted at room temperature for 1 hour. After sufficient washing, absorbance (450 nm) was measured with a plate reader using a peroxidase coloring kit (S-Bio SUMILON). The maximum dilution factor showing the absorbance value of 2 times or more of the yolk antibody of each bird species before immunization was shown as the ELISA value. The results are shown in Table 4.

By immunizing Pseudomonas aeruginosa homogenate, it was found that highly sensitive egg yolk antibodies against not only Pseudomonas aeruginosa (bacteria) but also ceramidase were produced in ostriches, chickens and quails. In particular, despite the immunization of each bird species with the same amount of antigen, giant ostriches produced the most reactive antibodies. This shows that if ostriches are used, highly sensitive antibodies can be produced with a small amount of antigen.

Ceramide plays an important role in moisturizing the skin and maintaining the barrier function, but is digested by endogenous ceramidase and the like. Ceramidase is an enzyme also produced from bacteria such as Pseudomonas aeruginosa, and is involved in infiltration of pathogenic bacteria into the skin by decomposing ceramide in the skin. It is considered that the inclusion of an antibody against ceramidase in cosmetics and pharmaceuticals can suppress the degradation of ceramide, enhance the skin barrier function and moisture retention, and normalize the skin function.

  The antibody according to the present invention can be contained not only in cosmetics and pharmaceuticals, but also in sterilizing sprays, fragrances and deodorants, and can also be contained in water used in humidifiers. The antibody according to the present invention may be carried on an air conditioner or a filter of a mask.

Claims (2)

  A composition for inhibiting ceramide degradation, comprising an antibody purified by inoculating an ostrich female with an antigen of Pseudomonas aeruginosa homogenate as an antigen, and purified from an egg laid by the ostrich female.   A cosmetic comprising the antibody of claim 1.